Cell growth inhibitor and screening method thereof

ABSTRACT

An object is to provide a cell growth inhibitor also effective for androgen-independent prostate cancer. The present invention provides a cell growth inhibitor having, as an active ingredient, an expression inhibitor or function inhibitor of PSF.

A computer readable text file, entitled “SequenceListing.txt,” createdon or about Mar. 28, 2013 with a file size about 75 kb contains thesequence listing for this application and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a growth inhibitor of cells, such asprostate cancer cells, in which expression of a predetermined antisenseRNA of a CTBP1 gene has been enhanced, a method of screening a cellgrowth inhibitor, and the like.

BACKGROUND ART

Prostate cancer is a cancer which manifests in men most frequently inEurope and United States. Also in Japan, with westernization of dietaryhabits and aging of population, the number of patients suffering fromprostate cancer is increasing drastically. Methods used widely for thetreatment of prostate cancer are surgical therapy includingprostatectomy, chemotherapy with an anticancer agent, and radiationtherapy. Surgical therapy is the first-line treatment, but when canceris diagnosed as being in an advanced stage or when surgery cannot beselected because it is a recurrent cancer after surgery, a therapeuticmethod other than surgery is selected.

In general, proliferation of prostate cancer is stimulated by androgen.Androgen is a steroid hormone having functions such as sexdifferentiation into male, function maintenance of reproductive organs,secondary sexual development, spermatogenesis, and promotion of anabolicaction in skeletal muscles and the like. Two androgens (testosterone anddihydrotestosterone (DHT) are mainly involved in masculinization ofhumans. Testosterone is, after synthesis in testicular interstitialcells, transported to target cells such as prostate cells via the bloodstream. In the cells, testosterone is converted into DHT by5-α-hydrogenase and the resulting DHT binds to an androgen receptor (AR)in the cytoplasm. AR bound to DHT becomes an active type, is transportedinto the nucleus, and binds to an androgen responsive sequence on thetarget gene to function as a transcription factor activating expressionof the target gene.

As therapeutic methods of prostate cancer other than surgical treatment,hormone therapy for inhibiting production and function of androgen isoften employed and in most cases, it produces considerably good effects.Within several years after this hormone therapy, however,androgen-independent prostate cancer sometimes occurs. It thereforebecomes an important object to control androgen-independent cancer.

Detailed molecular mechanism how androgen-dependent cancer progresses toandrogen-independent cancer has not yet been elucidated, but involvementof AR in it has been suggested. More specifically, it has been suggestedthat in androgen independent cancer, AR which has undergone mutation oramplification shows sensitivity to ultra-low-concentration androgen oranother steroid hormone (refer to, for example, Non-patent Documents 1to 3). A method of treating prostate cancer by inhibiting binding of ARto ligand or inhibiting expression or function of AR by RNAi (RNAinterference) method has been studied (refer to, for example, Non-patentDocuments 4 to 6).

There is however a limitation in the method of inhibiting the functionor expression of AR particularly for the treatment of prostate cancerwhich has recurred and a clinically sufficient method has not yet beendeveloped.

PRIOR ART DOCUMENTS Non-Patent Documents

-   Non-patent Document 1: Suzuki H, et al. Endocr. Relat. Cancer 10,    2003, 209-216-   Non-patent Document 2: Chen C D, et al. Nat. Med. 10, 2004, 33-39-   Non-patent Document 3: Debes J D, et al. N Eng J Med 351, 2004,    1488-1490-   Non-patent Document 4: Sun A, et al. Int J. Cancer. 2009 Jul. 29.    [Epub ahead of print] PMID: 19642108-   Non-patent Document 5: Compagno D, et al. PLoS One. 2007 Oct. 10;    2(10): e1006.PMID: 17925854-   Non-patent Document 6: Snoek R, et al. Clin Cancer Res. 2009 Jan. 1;    15(1): 39-47

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide a cell growth inhibitoralso effective for androgen-independent prostate cancer.

Means for Solving the Problem

With a view to overcoming the above-mentioned problem, the presentinventors thought that since inhibition of AR was an importanttherapeutic method of prostate cancer and in androgen-independent cancerfor which endocrine treatment was ineffective, AR signals were activatedby mutation or amplification of AR, signals downstream of AR were usefulas a therapeutic target. Based on this thought, an AR binding sites anda histone H3 acetylation sites were identified by using the chromatinimmunoprecipitation method and a human whole genome tiling array incombination (ChIP-chip method; refer to, for example, Cawley S, et al.Cell 116, 2002, 499-509, Kaneshiro K, et al. Genomics. 89, 2007,178-188) and moreover, by cap analysis gene expression (CAGE method,refer to, for example, Shiraki T, et al. Proc Natl Acad Sci USA. 100,2003, 15776-81, FANTOM Consortium, Nat. Genet. 41, 2009, 553-562),transcriptional start sites were genome-widely identified and thepromoter usage was profiled.

As a result, it has been found that a CTBP1 protein known to function asa corepressor in the nucleus functions as a transcription inhibitor ofAR. It has been found during the study on CTBP1 that a transcript of asense DNA in the vicinity of the AR binding site of the CTBP1 gene, thatis, an antisense RNA in the vicinity of the AR binding site of the CTBP1gene is induced strongly by androgen.

It has also been confirmed that suppression of the function of theantisense RNA by the RNAi enhances CTBP1 expression at both mRNA leveland protein level, while it suppresses transcriptional activity of ARand suppresses proliferation of prostate cancer cells.

It has also been confirmed that the expression amount of the antisenseRNA is significantly high in prostate cancer and metastatic cancer cellsthereof compared with that of normal prostate and the expression amountof CTBP1 is significantly low in prostate cancer and metastatic cancercells thereof compared with that of normal prostate.

It has further been confirmed that in a nude-mouse prostate cancer celltransplant model, the antisense RNA controls CTBP1 negatively andadministration of a double-stranded RNA having an RNAi effect againstthe antisense RNA suppresses proliferation of prostate cancer.

Moreover, it has been confirmed that with regard to a mechanism ofsuppressing CTBP1-AS and thereby suppressing the growth of prostatecancer, PSF which is an RNA-binding transcriptional repressor binds toCTBP1-AS and this complex suppresses transcription of a cellcycle-inhibiting gene to promote cell growth; the complex suppresses theexpression of CTBP1, resulting in promotion of transcription activationby AR; and accordingly, suppression of the expression of CTBP1-AS leadsto inhibition of cell proliferation and suppression of activation of AR.

Furthermore, the present inventors have confirmed that suppression ofPSF expression also results in inhibition of cell proliferation inprostate cancer, and completed the present invention.

The present invention therefore relates to:

[1] a cell growth inhibitor, which inhibits the growth of cells in whichany of the following antisense RNAs (which will hereinafter be called“CTBP1-AS”):

(i) an antisense RNA of a CTBP1 gene containing a partial sequence ofthe base sequence represented by SEQ ID NO:1;

(ii) an antisense RNA of a CTBP1 gene containing the base sequencerepresented by SEQ ID NO:5; and

(iii) a mutant or variant of the antisense RNA described above in (i) or(ii) or an antisense RNA in which one or several bases have beendeleted, added, or substituted in the antisense RNA described above in(i) or (ii)

has been expressed, containing a CTBP1-AS expression inhibitor orfunction inhibitor as an active ingredient;

[2] the cell growth inhibitor as described in claim 1, wherein theCTBP1-AS is an antisense RNA having a base sequence described in any ofSEQ ID NOS: 17 to 20;

[3] the cell growth inhibitor as described above in [1] or [2], whereinthe expression inhibitor or function inhibitor is a compound selectedfrom the group consisting of the following (a) to (d);

(a) antisense nucleic acids against CTBP1-AS or a portion thereof,

(b) nucleic acids having ribozyme activity to specifically cleaveCTBP1-AS;

(c) double-stranded nucleic acids having an RNAi effect againstCTBP1-AS; and

(d) nucleic acids encoding the nucleic acids described in any of (a) to(c);

[4] the cell growth inhibitor as described above in [3], wherein theexpression inhibitor or function inhibitor is a double-stranded RNAhaving an RNAi effect against CTBP1-AS and one strand of thedouble-stranded RNA has a sequence complementary to consecutive 19 to 30bases in CTBP1-AS;

[5] the cell growth inhibitor as described above in [3], wherein theexpression inhibitor or function inhibitor is a double-stranded RNAhaving an RNAi effect against CTBP1-AS and one strand of thedouble-stranded RNA has a base sequence complementary to the basesequence represented by any of SEQ ID NOS:3 to 6 and 24 to 52;

[6] the cell growth inhibitor as described above in any one of [1] to[5], wherein the cells in which CTBP1-AS has been expressed are prostatecancer cells and/or metastatic cancer cells thereof;

[7] a pharmaceutical composition containing the cell growth inhibitor asdescribed above in any one of [1] to [6];

[8] a method of preventing or treating prostate cancer, which includesadministering a therapeutically effective amount of the cell growthinhibitor as described above in any one of [1] to [6];

[9] a method of screening growth inhibitors of cells in which CTBP1-AShas been expressed, including a step of bringing cells in which CTBP1-AShas been expressed or a cell extract thereof into contact with testcompounds in the presence of androgen; a step of measuring an expressionlevel of CTBP1-AS; and a step of selecting, from the test compounds,test compounds decreasing the expression level of CTBP1-AS compared withan expression level measured in the absence of the test compounds;

[10] a method of screening proliferation inhibitors of cells in whichCTBP1-AS has been expressed, including a step of bringing cells whichhave expressed a CTBP1 gene or a cell extract thereof into contact withtest compounds in the presence of androgen; a step of measuring anexpression level of the CTBP1 gene; and a step of selecting, from thetest compounds, test compounds increasing the expression level of theCTBP1 gene compared with an expression level measured in the absence ofthe test compounds;

[11] the method as described above in [9] or [10], wherein the cells inwhich CTBP1-AS has been expressed are prostate cancer cells and/ormetastatic cancer cells thereof;

[12] a testing method for judging the prognosis of prostate cancer,including a step of measuring the expression level of CTBP1-AS in cellssampled from the prostate of a patient and a step of comparing theexpression level with an expression level in normal prostate cells; and

[13] a testing method for judging the prognosis of prostate cancer,including a step of measuring the expression level of a CTBP1 gene incells sampled from the prostate of a patient and a step of comparing theexpression level with an expression level in normal prostate cells.

The present invention further relates to:

[14] A cell growth inhibitor, which inhibits the growth of cells inwhich any of the following antisense RNAs (which will hereinafter becalled “CTBP1-AS”):

(i) an antisense RNA of a CTBP1 gene containing a partial sequence ofthe base sequence represented by SEQ ID NO:1;

(ii) an antisense RNA of a CTBP1 gene containing the base sequencerepresented by SEQ ID NO:5; and

(iii) a mutant or variant of the antisense RNA described above in (i) or(ii) or an antisense RNA in which one or several bases have beendeleted, added, or substituted in the antisense RNA described above in(i) or (ii)

has been expressed, comprising a PSF expression inhibitor or functioninhibitor as an active ingredient;

[15] The cell growth inhibitor according to [14] above, wherein theCTBP1-AS is an antisense RNA having a base sequence described in any ofSEQ ID NOS: 17 to 20;

[16] The cell growth inhibitor according to [14] above, wherein theexpression inhibitor or function inhibitor is a compound selected fromthe group consisting of the following (a) to (d);

(a) antisense nucleic acids against PSF transcripts or a portionthereof,

(b) nucleic acids having ribozyme activity to specifically cleave PSFtranscripts;

(c) double-stranded nucleic acids having an RNAi effect against PSFtranscripts; and

(d) nucleic acids encoding the nucleic acids described in any of (a) to(c);

[17] The cell growth inhibitor according to [16] above, wherein theexpression inhibitor or function inhibitor is a double-stranded RNAhaving an RNAi effect against PSF transcripts and one strand of thedouble-stranded RNA has a sequence represented by any of SEQ ID NOS:119to 126;

[18] The cell growth inhibitor according to any one of [14] to [17]above, wherein the cells in which CTBP1-AS has been expressed areprostate cancer cells and/or metastatic cancer cells thereof;

[19] A pharmaceutical composition comprising the cell growth inhibitoras claimed in any one of [14] to [17] above;

[20] A method of preventing or treating prostate cancer, comprisingadministering a therapeutically effective amount of the cell growthinhibitor as claimed in any one of [14] to [17] above.

Effect of the Invention

The cell growth inhibitor of the present invention can efficientlyinhibits the growth of cells in which expression of CTBP1-AS has beenenhanced such as prostate cancer cells by inhibiting the expression orfunction of CTBP1-AS or PSF.

The cell growth inhibitor of the present invention suppressestranscriptional activity of signals downstream of AR by inhibitingexpression or function of CTBP1-AS or PSF so that it is presumed to beeffective against androgen-independent cancer in which AR signals havebeen activated by AR variation or amplification.

Using a double-stranded nucleic acid having an RNAi effect againstCTBP1-AS or PSF as a cell growth inhibitor of the present inventionenables high target specificity and high safety, because it makes use ofa mechanism which cell originally have.

In addition, the present invention provides a screening method capableof efficiently selecting, from test compounds, a growth inhibitor ofcells in which expression of CTBP1-AS has been enhanced; a method ofpreventing or treating prostate cancer; and a testing method forevaluating the prognosis of prostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows measurement results of the expression amount of CTBP1-ASwhen siRNA against CTBP1-AS or a control double-stranded RNA wasadministered to LNCaP cells. The CTBP1-AS expression was induced byandrogen (R1881) and suppressed by the administration of siRNA againstCTBP1-AS.

FIG. 2 shows measurement results of the expression amount of CTBP1 (mRNAlevel) when siRNA against CTBP1-AS or a control double-stranded RNA wasadministered to LNCaP cells. The CTBP1 expression was suppressed byandrogen and the suppressive effect by androgen was decreased by theadministration of siRNA against CTBP1-AS.

FIG. 3 shows measurement results of the expression amount of CTBP1(protein level) when siRNA against CTBP1-AS or a control double-strandedRNA was administered to LNCaP cells. The CTBP1-AS expression wassuppressed by androgen and the suppressive effect by androgen wasdecreased by the administration of siRNA against CTBP1-AS.

FIG. 4A shows the results of experiments to confirm the influence ofCTBP1-AS on the transcriptional activity of AR obtained by introducing aPSA luciferase vector into LNCaP cells and administering siRNA againstCTBP1-AS or a control double-stranded RNA to the resulting cells.Luciferase activity, that is, transcriptional activity of AR wasincreased by androgen and suppressed by the administration of siRNAagainst CTBP1-AS.

FIG. 4B is studying results of the expression of an androgen responsivegene FKBP5 in the cells. The expression amount of the androgenresponsive gene was increased by androgen, but suppressed by theadministration of siRNA.

FIG. 4C is studying results of the expression of an androgen responsivegene TMPRSS2 in the cells. The expression amount of the androgenresponsive gene was increased by androgen, but suppressed by theadministration of siRNA.

FIG. 5 shows the results of experiments to investigate the influence offunction suppression of CTBP1-AS on the proliferation of LNCaP cells byusing MTS assay. Administration of siRNA against CTBP1-AS suppressedcell proliferation.

FIG. 6 shows the results of experiments to confirm the influence offunction suppression of CTBP1-AS on a tumor volume in a mouse tumormodel. Proliferation of tumor cells subcutaneously transplanted to micewas markedly suppressed by the administration of siRNA against CTBP1-AS.

FIG. 7 shows the results of experiments to confirm the influence offunction suppression of CTBP1-AS on a tumor weight in a mouse tumormodel. Proliferation of tumor cells subcutaneously transplanted to micewas markedly suppressed by the administration of siRNA against CTBP1-AS.

FIG. 8 shows the measurement results of the RNA level expression amountsof CTBP1-AS and CTBP1 in mouse tumor model. Administration of siRNAagainst CTBP1-AS decreased the expression amount of CTBP1-AS in tumorbut increased the expression amount of CTBP1.

FIG. 9 shows the measurement results of the protein level expressionamounts of CTBP1 in mouse tumor model. Administration of siRNA againstCTBP1-AS increased the expression amount of the CTBP1 protein in tumor.

FIG. 10 shows the comparison results of the expression amount amongnormal, cancer, and metastatic cancer samples determined by extractingthe signal intensity of a probe specific to AX747592 based on thepublished expression profile data. The expression amount of CTBP1-AS incancer and metastasis cancer tissues was significantly higher than thatin normal prostate.

FIG. 11A shows the results of experiments to assess the CTBP1 expressionin prostate cancer cells by using immunostaining method. The CTBP1expression amount was decreased in prostate cancer cells compared withthat in normal cells.

FIG. 11B shows the comparison results of a survival rate between CTBP1high expression group and low expression group. The survival rate wassignificantly higher in the CTBP1 high expression group than that in thelow expression group.

FIG. 11C shows images of typical sections of normal prostate andprostate cancer obtained by immunostaining method.

FIG. 12 is a schematic view showing sequences of CTBP1-ASa, CTBP1-ASb,and CTBP1-ASd whose existence has been confirmed as a result ofsearching for CTBP1-ASs other than CTBP1-ASd by using the 3′ RACE PCRmethod.

FIG. 13 shows the northern blotting results by which expression ofCTBP1-AS was confirmed using LNCaP-cell-derived RNA. An about 5-kb longtranscription product is detected, suggesting that CTBP1-ASb isexpressed mainly.

FIG. 14 shows the results of analyzing localized CTBP1-AS by northernblotting using nuclear RNA and cytoplasmic RNA (FIG. 14 a) and theresults of measuring the expression amount of cDNA synthesized from RNAby using qRT-PCR (FIG. 14 b, corrected by GAPDH).

FIGS. 15 a and 15 b show the analysis results of the influence ofandrogen stimulation on the expression of CTBP1 and CTBP1-AS in LNCaPcells and LTAD cells which will be an androgen depletion resistant modelby western blotting and northern blotting. FIGS. 15 c and 15 d showcomparison results of cell proliferation ability compared by suppressingCTBP1-AS expression by RNAi method with or without androgen stimulationin LNCaP cells or LTAD cells.

FIG. 16 shows the measurement results of a tumor volume (b), mRNA-levelCTBP1-AS expression amount (c), and protein-level CTBP1 expressionamount (d), after transplanting LTAD cells of an androgen-depletionresistant model to nude mice and locally injecting siRNA againstCTBP1-AS.

FIG. 17 shows the results of experiments to confirm the involvement ofPSF, an RNA-binding transcriptional repressor, in expression suppressionof CTBP1. When transcription of PSF was suppressed by RNAi, thesuppression degree of CTBP1 expression by androgen stimulationdecreased.

FIG. 18 shows immunoprecipitation results showing androgen-dependentbinding of PSF to CTBP1-AS.

FIG. 19 shows the results of analyzing localization of PSF and CTBP1-ASby RNA-FISH. Localization of PSF almost coincided with that of CTBP1-AS.

FIG. 20 shows the results of analyzing the relationship between PSF andcell cycle.

FIG. 21 shows the results of analyzing PSF-mediated control of SMAD3 andp53 by using siRNA against PSF through qRT-PCR (a) and western blotting(b).

FIG. 22 shows the results of studying the control of a cellcycle-related factor by CTBP1-AS by using siRNA against CTBP1-AS.

FIG. 23 is an explanatory view showing a gene expression controlmechanism by CTBP1-AS and PSF.

FIG. 24 shows the results of studying the CTBP1-AS expressionsuppression effect of 26 siRNAs.

FIG. 25 shows the results of experiments to determine the expressionamount of PSF after each siRNA against PSF or control siRNA (siControl)has been introduced into LNCaP cells.

FIG. 26 shows the results of MTS assay after each siRNA against PSF orcontrol siControl has been introduced into LNCaP cells.

FIG. 27 shows the results of western blotting to determine theexpression amount of PSF protein after each siRNA against PSF orsiControl has been introduced into LNCaP cells.

FIG. 28 shows the picture of mouse prostate cancer xenograft models fiveweeks after the administration of siPSF or siControl.

FIG. 29 shows the change of tumor volume in the prostate cancerxenograft models after the administration of siPSF or siControl.

MODE FOR CARRYING OUT THE INVENTION

<Cell Growth Inhibitor Containing a CTBP1-AS Expression Inhibitor orFunction Inhibitor>

The cell growth inhibitor according to the present invention ischaracterized in that it suppresses proliferation of cells in whichCTBP1-AS has been expressed and that it contains a CTBP1-AS expressioninhibitor or function inhibitor as an active ingredient.

The term “CTBP1” as used herein means a C-terminal binding protein 1 andthe term “CTBP1 gene” means a gene encoding a CTBP1 protein. CTBP1 isknown to function as a corepressor in the nucleus (Kim J H, et al. NatStruct Mol. Biol. 12, 2005, 423-428, Senyuk V, et al. Arch BiochemBiophys. 441, 2005, 168-173). For example, involvement of CTBP1 intranscription suppression of a ZEB1 gene and the like has been reportedand generally, it is presumed to negatively control the transcription(Chinnadurai G, Int J Biochem Cell Biol. 39, 2007, 1593-1607).

As described later in Examples, the present inventors have found that inprostate cancer cells, expression of CTBP1 is significantly reduced andit is controlled by androgen in both mRNA level and protein levelexpression.

The term “CTBP1-AS” as used herein means any one of antisense RNAs (i)to (iii) of a CTBP1 gene expressed in the vicinity of the AR bindingsite of the CTBP1 gene.

(i) An antisense RNA of a CTBP1 gene containing a partial sequence ofthe base sequence represented by SEQ ID NO:1,

(ii) an antisense RNA of the CTBP1 gene containing the base sequencerepresented by SEQ ID NO:5, and

(iii) a mutant or variant of the antisense RNA described above in (i) or(ii) or an antisense RNA in which one or several bases have beendeleted, added, or substituted in the antisense RNA described above in(i) or (ii).

The term “antisense RNA” means a transcription product formed with asense chain (sense DNA) of a double-stranded DNA of a gene as atemplate.

CTBP1-AS is a molecule which will be a target of the cell growthinhibitor according to the present invention. In the living body, thereis a plurality of antisense RNAs capable of satisfying any one of theabove (i) to (iii) and all of them will become CTBP1-AS. The antisenseRNAs (i) to (iii) will next be described respectively in detail.

(i) Antisense RNA Of a CTBP1 Gene Containing a Partial Sequence of theBase Sequence Represented by SEQ ID NO:1

The antisense RNA of a CTBP1 gene having a base sequence represented bySEQ ID NO:1 is a transcription product of a sense DNA in the vicinity ofthe AR binding site of the CTBP1 gene and its cDNA is known to haveAccession Number: AX747592 (SEQ ID NO:2).

Examples of the antisense RNA of a CTBP1 gene containing a partialsequence of the base sequence represented by SEQ ID NO:1 include:

an antisense RNA consisting of only a partial base sequence of the basesequence represented by SEQ ID NO:1;

an antisense RNA comprising a partial sequence containing the 5′-end ofthe base sequence represented by SEQ ID NO:1 and further having one ormore bases bound to the 5′-end; and

an antisense RNA comprising a partial sequence containing the 3′ end ofthe base sequence represented by SEQ ID NO:1 and further having one ormore bases bound to the 3′ end.

The antisense RNAs expressed in the living body correspond to theantisense RNA of (i) insofar as it contains a partial sequence of thebase sequence represented by SEQ ID NO:1. As a target of the cell growthinhibitor of the present invention, the antisense RNA consisting of apartial sequence containing the 3′ end of the base sequence representedby SEQ ID NO:1 and the antisense RNA containing a partial sequencecontaining the 3′ end of the base sequence represented by SEQ ID NO:1and having one or more bases bound to the 3′ end are particularlypreferred. Examples of the “partial sequence containing the 3′ end ofthe base sequence represented by SEQ ID NO:1” include, but not limitedto, a partial sequence from position 1300 to the 3′ end of the basesequence described in SEQ ID NO:1, a partial sequence from position 1200to the 3′ end, and a partial sequence from position 800 to the 3′ end.

(ii) Antisense RNA of the CTBP1 Gene Containing the Base SequenceRepresented by SEQ ID NO: 5.

Examples of the antisense RNA of the CTBP1 gene containing the basesequence represented by SEQ ID NO:5 include an antisense RNA consistingonly of the base sequence represented by SEQ ID NO:5 and an antisenseRNA having one or more bases bound to the 3′ end and/or 5′ end of thebase sequence described in SEQ ID NO:5.

The base sequence represented by SEQ ID NO:5 corresponds to fromposition 2348 to position 2372 of the base sequence described in SEQ IDNO:1. The number of bases bound to the 3′ end and/or 5′ end is notparticularly limited. The present inventors have confirmed that as shownlater in Examples, administration of siRNA to this region as a targetinhibits transcriptional activity of AR and also inhibits proliferationof prostate cancer cells. Accordingly, it is obvious that an expressioninhibitor or function inhibitor of CTBP1-AS containing the base sequencerepresented by SEQ ID NO:5 is suited as an active ingredient of the cellgrowth inhibitor of the present invention.

As an example of the antisense RNA (ii), an antisense RNA containing thebase sequence described in SEQ ID NO:23 is also preferred.

(Iii) Mutant or Variant Of the Antisense RNA as Described Above in (i)or (ii) or an Antisense RNA in which One or Several Bases have beenDeleted, Added, or Substituted in the Antisense RNA Described in (i) or(ii).

Examples of the mutant or variant of the antisense RNA described in (i)or (ii) include various variants including splicing variants such as 5′alternative splicing and 3′ alternative splicing, polymorphisms such assingle nucleotide polymorphism (SNP) and copy number variation (CNV),and mutants (for example, cancer-derived mutants).

Also these mutants or variants can be expressed as a transcriptionproduct of a sense DNA in the vicinity of the AR binding site of a CTBP1gene and will be a target of the cell growth inhibitor according to thepresent invention.

Although no particular limitation is imposed on the number of basesdeleted or the like from the antisense RNA described in (i) or (ii) inwhich one or several bases have been deleted, added, or substitutedinsofar as it is a transcription product of a sense DNA in the vicinityof the AR binding site of the CTBP1 gene. The number is from 1 to 10,preferably from 1 to 5, more preferably from about 1 to 3 or correspondsto within 10%, preferably within 5%, more preferably within 1% of theentire length. Also the position of the bases to be deleted or the likeis not particularly limited and it may be the base(s) at the 5′ end or3′ end of the antisense RNA or the base(s) other than that at the ends.Alternatively, bases may be deleted, added, and/or substituted at aplurality of positions.

Such antisense RNAs may be expressed as a transcription product of thesense DNA at the AR binding site of CTBP1 gene and will be a target ofthe cell growth inhibitor according to the present invention.

Specific examples of the CTBP1-AS include RNAs consisting of the basesequence described in SEQ ID NOS:17 to 21.

The RNA having the base sequence described in SEQ ID NO:17 is an RNA of3710 bases in total length which contains from position 698 to the 3′end of the base sequence described in SEQ ID NO:1 and further has an RNAof 522 bases bound to the 3′ end (which will hereinafter be called“CTBP1-ASc”).

The RNA having the base sequence described in SEQ ID NO:18 is an RNA of5041 bases in total length which contains from position 698 to the 3′end of the base sequence described in SEQ ID NO:1 and further has an RNAof 1853 bases bound to the 3′ end (which will hereinafter be called“CTBP1-ASb”).

The RNA having the base sequence described in SEQ ID NO:19 is an RNA of15756 bases in total length which contains from position 698 to the 3′end of the base sequence described in SEQ ID NO:1 and further has an RNAof 12568 bases bound to the 3′ end (which will hereinafter be called“CTBP1-ASa”).

The RNA having the base sequence described in SEQ ID NO:20 is an RNA of3189 bases in total length corresponding to from position 697 to the 3′end of the base sequence described in SEQ ID NO:1 (which willhereinafter be called “CTBP1-ASd”).

It has already been confirmed that any of these antisense RNAs has beenexpressed as a transcription product of a sense DNA in the vicinity ofthe AR binding site of a CTBP1 gene so that they will be a target of thecell growth inhibitor according to the present invention.

The present inventors have recently found newly by analysis usingprostate cancer cells LNCaP that expression of CTBP1-AS is stronglyinduced by androgen. CTBP1-AS is a non-coding RNA which does not encodea protein motif. CTBP1-AS has an enhanced expression level in prostatecancer cells. Inhibition of CTBP1-AS by the RNAi method leads tosuppression of transcriptional activity of AR and remarkable inhibitionof cancer cell growth.

As will be described later in Examples, when CTBP1-AS binds to PSF,which is an RNA-binding transcriptional repressor, to form a complex,the resulting complex suppresses transcription of a cell-cycleinhibiting gene (for example, SMAD3 and p53). As a result, cellproliferation is accelerated.

On the other hand, the complex between CTBP1-AS and PSF also suppressestranscription of CTBP1 in cooperation with a histone deacetylationenzyme. Since CTBP1 originally has a function of suppressingtranscription of an androgen receptor, suppression of transcription ofCTBP1 enhances transcription activation by the androgen receptor. Thisleads to cell proliferation.

Accordingly, when the expression of CTBP1-AS is suppressed, as will beshown later in Examples, the proliferation of androgen-resistant cancercells can also be suppressed.

The term “inhibition of cell growth” means that the proliferation ofcells can be terminated or retarded.

The cell growth inhibitor according to the present invention iseffective for all the cells in which CTBP1-AS has been expressed, but itis particularly useful for suppressing proliferation of cells showingenhanced CTBP1-AS expression. The term “cells showing enhanced CTBP1-ASexpression” as used herein means cells having a significantly increasedCTBP1-AS expression level compared with normal non-dividing cells.Examples of the cells having enhanced CTBP1-AS expression include, butnot limited to, prostate cancer cells and metastatic cancer cellsthereof. The term “metastatic cancer cells of prostate cancer” meanscancer cells found in metastasis of prostate cancer.

The term “CTBP1-AS expression inhibitor” as used herein means asubstance completely inhibiting transcription of CTBP1-AS, that is,transcription for production of CTBP1-AS using the DNA of a CTBP1 geneas a template or a substance significantly reducing such transcription.The term “expression” as used herein embraces transcription forproduction of a complementary RNA by using a DNA as a template and atranscriptase, synthesis of a protein based on genetic information whicha DNA has, and translation for synthesis of a protein based on geneticinformation which mRNA has.

The term “CTBP1-AS function inhibitor” as used herein means a substanceacting on CTBP1-AS itself and completely inhibiting its function or asubstance significantly reducing the function. Examples include, but notlimited to, substances promoting degradation of CTBP1-AS and substanceshybridized with CTBP1-AS. The term “function of CTBP1-AS” as used hereinmeans a function of suppressing expression of CTBP1 and activating ARsignals downstream. Inhibition of the function of CTBP1-AS thereforemeans enhancing CTBP1 expression and suppressing activation of ARsignals downstream.

The CTBP1-AS expression inhibitor or function inhibitor to be used inthe present invention may be any substance insofar as it has theabove-described function. Examples include low molecular compounds, highmolecular compounds, proteins including antibodies and enzymes, andnucleic acids including single-stranded DNAs, double-stranded DNAs,single-stranded RNAs, double-stranded RNAs, chimeric nucleic acidscontaining a single-stranded or double-stranded DNA and asingle-stranded or double-stranded RNA, and artificial nucleic acids(peptide nucleic acids (PNA) and locked nucleic acids (LNA)).

As the CTBP1-AS expression inhibitor or function inhibitor, thefollowing ones are preferably used:

(a) antisense nucleic acids against CTBP1-AS or a portion thereof,

(b) nucleic acids having ribozyme activity to specifically cleaveCTBP1-AS,

(c) double-stranded nucleic acids having an RNAi effect againstCTBP1-AS, and

(d) nucleic acids capable of expressing the nucleic acid described inany one of (a) to (c).

(a) Antisense Nucleic Acids Against CTBP1-AS or a Portion Thereof

The antisense nucleic acid method is well known as an expressioninhibiting method. It is a method of introducing a single-strandednucleic acid (antisense nucleic acid) having a base sequencecomplementary to a target gene (basically, mRNA which is a transcriptionproduct), hybridizing it with the target gene, and thereby inhibitinggene expression.

The antisense nucleic acid to be used in the present invention haspreferably a sequence complementary to a portion of CTBP1-AS but it isnot necessary that the sequence is completely complementary. Forexample, 90% or more, preferably 95% or more, more preferably 98% ormore of the antisense nucleic acid is complementary to a portion ofCTBP1-AS. No particular limitation is imposed on the length of theantisense nucleic acid insofar as it can produce an effect of inhibitingthe function of CTBP1-AS. It has usually a length of from 10 bases to100 bases, preferably a length of from 15 to 30 bases.

The antisense nucleic acid can be designed and synthesized as neededbased on the base sequence of CTBP1-AS by those skilled in the art byusing known software or the like. The antisense nucleic acid may be anyof DNA, RNA, and chimeric nucleic acids containing DNA and RNA. They maybe modified. Examples of the modified nucleic acid include thoseobtained by substituting a phosphate group with a thiophosphate group ora methyl phosphate group.

(b) Nucleic Acids Having Ribozyme Activity to Specifically CleaveCTBP1-AS

Examples of the CTBP1-AS function inhibitor also include nucleic acidshaving ribozyme activity to specifically cleave CTBP1-AS. Ribozyme is anucleic acid molecule catalytically hydrolyzing a target RNA and iscomposed of an antisense region having a sequence complementary to atarget RNA and a catalytic center region involved in cleavage reaction(for example, Ribozyme: Biochemistry and Biotechnology (Krupp, G. &Gaur, R. K. eds: Eaton Publishing, MA, 2000). Ribozyme whichspecifically cleaves CTBP1-AS can also be designed as needed in a mannerknown by those skilled in the art.

Ribozyme is usually an RNA molecule but a chimeric DNA-RNA molecule canalso be used.

(c) Double-Stranded Nucleic Acids Having an RNAi Effect Against CTBP1-AS

As the CTBP1-AS function inhibitor, double-stranded nucleic acids havingan RNAi effect against CTBP1-AS are also preferred. RNAi is asequence-specific gene expression suppressing mechanism induced by adouble-stranded nucleic acid. It has very high target specificity and ishighly safe because it utilizes a gene expression suppressing mechanismoriginally present in the living body.

When it is used for mammal cells, a small double-stranded RNA (smallinterference RNA; siRNA) usually with from about 19 to 30 bases,preferably from about 21 to 25 bases is used. A longer double-strandedRNA which will be cleaved in the cell by an enzyme (Dicer) and becomesiRNA may also be used.

Cleavage of a target RNA molecule by RNAi is conducted as follows.First, siRNA is single-stranded by one-side chain cleavage by a proteincalled Argonaute or by rewinding by RNA helicase and then it binds toArgonaute to form a RISC(RNA induced silencing complex) which is aneffector complex. In the RISC, siRNA serves as a guide moleculesearching for the target RNA, while Argonaute functions as aribonuclease (Slicer) cutting the target RNA.

A siRNA has typically a completely complementary double-stranded regionof 19 bases and has two protruding bases (overhangs) at the 3′ end ofeach of the sense strand and antisense strand but it may be a blunt endtype having no overhangs. For example, a blunt end RNA with 25 bases isadvantageous because it minimizes the activation of an interferonresponsive gene, prevents an off-target effect derived from the sensestrand, and has considerably high stability in the serum so that it issuited for use also in vivo.

Judging from the sequence of CTBP1-AS, the siRNA to be used in thepresent invention can be designed, by a known method for obtaining siRNAwith high activity, by selecting a target region not having a sequencesimilar to that of another gene, containing a consensus sequence ofsiRNA with high activity, having a periodicity of three bases in itssequence, and having a GC content of from about 35 to 45%. For designingof siRNA, a known activity prediction algorithm can also be used. Assuch an activity prediction algorithm, siExplorer, siDirect, BIOPREDsi,and the like have been made public.

The double-stranded nucleic acid having an RNAi effect may be adouble-stranded RNA or a chimeric DNA-RNA double-stranded nucleic acid,or it may contain an artificial nucleic acid. The chimeric type isobtained by substituting a portion of the double-stranded RNA having anRNAi effect with DNA so that it is known to have high stability in theserum and a low immunoresponse induction property.

The above-described double-stranded nucleic acid can have improvedresistance to nuclease or be made more stable by modification of the2′-OH group, phosphorothioate backbone substitution, modification with aboranophosphate group, or introduction of LAN (locked nucleic acid)having ribose bridged between the 2 and 4 positions thereof. Such amodified double-stranded nucleic acid is also embraced in the presentinvention.

(d) Nucleic Acids Capable of Expressing the Nucleic Acid Described Abovein any One of (a) to (c)

The nucleic acids described above in (a) to (c) can be administered tothe living body after synthesis. It is also possible to introduce anucleic acid encoding any of these nucleic acids (a) to (c) into theliving body and express the nucleic acid in cells.

For example, when a vector containing a DNA capable of expressing anantisense nucleic acid by transcription or a DNA capable of expressing anucleic acid having ribozyme activity by transcription is introducedinto cells, RNA expressed in the cells can inhibit the function ofCTBP1-AS.

Further, a vector containing DNAs encoding the respective strands of adouble-stranded RNA having an RNAi effect against CTBP1-AS may beintroduced into cells. These two strands are expressed respectively andhybridized in the cells and as a result, produce an RNAi effect againstCTBP1-AS. Alternatively, in order to express a double-stranded RNAhaving an RNAi effect, a vector containing a DNA encoding asingle-stranded RNA obtained by binding respective strands of thedouble-stranded RNA via a loop can also be used. The single-stranded RNAthus expressed is hybridized in the molecule, constitutes a shorthairpin RNA, is cleaved with Dicer at the loop structure portionthereof, and becomes an siRNA.

A construct capable of expressing a desired nucleic acid in cells can bedesigned as needed by those skilled in the art and introduced into thecells.

Examples of the double-stranded nucleic acids (c) having an RNAi effectagainst CTBP1-AS include those that target regions, in the base sequenceof CTBP1-AS represented by SEQ ID NO:1, from position 1804 to position1828 (SEQ ID NO:3), from positions 1894 to position 1918 (SEQ ID NO:4),from position 2348 to position 2372 (SEQ ID NO:5), and from position3063 to position 3087 (SEQ ID NO:6).

Examples of the double-stranded nucleic acids targeting them includethose having, as one of two strand, an RNA having a base sequencecomplementary to these targets and, as the other strand, an RNA having abase sequence complementary to the former one. Such double-strandednucleic acids are shown in the following table.

TABLE 1 Abbreviation Target siRNA siRNA[1] 1804-1828UGACCAGUCCGUUUGACACUGAGUG (SEQ ID NO. 7)CACUCAGUGUCAAACGGACUGGUCA (SEQ ID NO. 8) siRNA[2] 1894-1918UUGAGAUGCCGGAAACAUUGAUGGG (SEQ ID NO. 9)CCCAUCAAUGUUUCCGGCAUCUCAA (SEQ ID NO. 10) siRNA[3] 2348-2372UUAUGUCUCCAGCAAGCUUGGUCUU (SEQ ID NO. 11)AAGACCAAGCUUGCUGGAGACAUAA (SEQ ID NO. 12) siRNA[4] 3063-3087GAGACAGGAGGAUGUAGUUUCUAAU (SEQ ID NO. 13)AUUAGAAACUACAUCCUCCUGUCUC (SEQ ID NO. 14)

The double-stranded nucleic acids of the present invention include thedouble-stranded nucleic acids having the above-described four regions asa target consisting of a strand having mismatches with the target RNA atfrom one to three bases and the other strand having a base sequencecomplementary to it, insofar as they produce an RNAi effect.

As the CTBP1-AS function inhibitor or expression inhibitor, an siRNA(for example, siRNA [3]) targeting a region represented by SEQ ID NO:5is particularly preferred.

Examples of the double-stranded nucleic acids (c) having an RNAi effectagainst CTBP1-AS include those that target the sequence of CTBP1-ASrepresented by SEQ ID NOS:24 to 52. Such double-stranded nucleic acidsinclude double-stranded nucleic acids having an RNA composed of a basesequence represented by SEQ ID NOS:53 to 78 and an RNA complementarythereto. Of these, nucleic acids Nos. 3, 4, 6, 10, 12, 18, and 24 arehighly effective for suppressing the expression of CTBP1-AS.

<Cell Growth Inhibitor Containing a PSF Expression Inhibitor or FunctionInhibitor>

The term “PSF” as used herein means a RNA-binding transcriptioninhibitor, also called as PSF/SFPQ (slicing factor, proline-glutaminerich). As described above, PSF binds to CTBP1-AS and forms a complexthat suppresses transcription of a cell-cycle inhibiting genes such asSMAD3 and p53, which results in acceleration of cell proliferation.

Furthermore, the complex of CTBP1-AS and PSF suppresses thetranscription of CTBP1 cooperatively with a histone deacetylationenzyme. Since CTBP1 has a function of suppressing transcription of anandrogen receptor, suppression of transcription of CTBP1 enhancestranscription activation by the androgen receptor. As a result, the cellgrowth is inhibited.

The inventors of the present application have found, as will be shownlater in Examples, that the suppression of PSF expression, like thesuppression of CTBP1-AS expression, leads to the suppression of thegrowth of androgen resistant cancer cells.

The cell growth inhibitor containing a PSF expression inhibitor orfunction inhibitor according to the present invention is effective forall the cells in which CTBP1-AS has been expressed, but it isparticularly useful for suppressing proliferation of cells showingenhanced CTBP1-AS expression.

The terms that are used in the descriptions of the cell growth inhibitorcontaining a PSF expression inhibitor or function inhibitor and are alsoused in the descriptions of the cell growth inhibitor containing aCTBP1-AS expression inhibitor or function inhibitor have the samemeaning as those used in the descriptions of the cell growth inhibitorcontaining a CTBP1-AS expression inhibitor, unless otherwise specified.

The term “expression” as used herein embraces transcription forproduction of a complementary RNA by using a DNA as a template and atranscriptase, and translation for synthesis of a protein based ongenetic information which mRNA has. The term “PSF expression inhibitor”means a substance that completely inhibits transcription or translation,or significantly reduces such transcription or translation.

The term “PSF function inhibitor” as used herein means a substanceacting on PSF itself and completely inhibiting its function or asubstance significantly reducing the function. Examples include, but notlimited to, substances promoting degradation of PSF and substanceshybridized with nucleic acids encoding PSF. The term “function of PSF”as used herein means a function of forming a complex with CTBP1-AS andactivating AR signals downstream. Inhibition of the function of PSFtherefore means enhancing CTBP1 expression and suppressing activation ofAR signals downstream.

The PSF expression inhibitor or function inhibitor to be used in thepresent invention may be any substance insofar as it has theabove-described function. Examples include low molecular compounds, highmolecular compounds, proteins including antibodies and enzymes, andnucleic acids including single-stranded DNAs, double-stranded DNAs,single-stranded RNAs, double-stranded RNAs, chimeric nucleic acidscontaining a single-stranded or double-stranded DNA and asingle-stranded or double-stranded RNA, and artificial nucleic acids(peptide nucleic acids (PNA) and locked nucleic acids (LNA)).

As the PSF expression inhibitor or function inhibitor, the followingones are preferably used:

(a) antisense nucleic acids against PSF transcripts or a portionthereof,

(b) nucleic acids having ribozyme activity to specifically cleave PSFtranscripts,

(c) double-stranded nucleic acids having an RNAi effect against PSFtranscripts, and

(d) nucleic acids capable of expressing the nucleic acid described inany one of (a) to (c).

Examples of (c) double-stranded nucleic acids having an RNAi effectagainst PSF transcripts include the siRNAs, one strand of which has asequence selected from the followings:

(SEQ ID NO: 119) GCACGUUUGAGUACGAAUAUU (SEQ ID NO: 120)GGCACGUUUGAGUACGAAUAU (SEQ ID NO: 121) GCAUAUUAGGCUACGUAUUCC(SEQ ID NO: 122) GAUGAUCGUGGAAGAUCUACA (SEQ ID NO: 123)GGGAGAUCCCUAUGGUUCAGG (SEQ ID NO: 124) GUUUGGGCAGGUAAAAUUAUG(SEQ ID NO: 125) CAUAGGUUAUGAAGCUAAUCC (SEQ ID NO: 126)GGCAUAGGUUAUGAAGCUAAU<Pharmaceutical Composition>

The pharmaceutical composition of the present invention contains theabove-mentioned cell growth inhibitor according to the presentinvention, i.e., (i) the cell growth inhibitor containing a CTBP1-ASexpression inhibitor or function inhibitor or (ii) the cell growthinhibitor containing a PSF expression inhibitor or function inhibitor.The pharmaceutical composition of the present invention administered tocells in which CTBP1-AS expression has been enhanced therefore producesa proliferation suppressive effect against these cells so that it isparticularly useful as a preventive or remedy of diseases in whichAR-dependent cell proliferation is involved. Examples of the diseases inwhich AR-dependent cell proliferation is involved include AR-dependentcancers (typically, prostate cancer) and benign prostatic hyperplasia.

The pharmaceutical composition of the present invention may contain apharmaceutically acceptable carrier or additive. Examples of such acarrier include, but not limited to, water, saline, phosphate buffer,dextrose, glycerol, pharmaceutically acceptable organic solvents such asethanol, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinylpolymer, carboxymethyl cellulose sodium, sodium polyacrylate, sodiumalginate, water-soluble dextran, carboxymethyl starch sodium, pectin,methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein,agar, polyethylene glycol, diglycerin, glycerin, propylene glycol,petrolatum, paraffin, stearyl alcohol, stearic acid, human serumalbumin, mannitol, sorbitol, lactose, surfactants, excipients, flavoringagents, preservatives, stabilizers, buffers, suspending agents, tonicityagents, binders, disintegrants, lubricants, fluidity accelerators, tastecorrigents, and the like.

The pharmaceutical composition of the present invention can beformulated into various forms, for example, liquids (such asinjections), dispersants, suspensions, tablets, pills, powders, andsuppositories. A preferred mode of the pharmaceutical composition is aninjection and it is preferably administered parenterally (for example,intravenously, transdermally, intraperitoneally, or intramuscularly).

When the pharmaceutical composition of the present invention contains,as a cell proliferation inhibitor, a nucleic acid such as that describedabove in (a) to (c), preparations can be obtained by enclosing thenucleic acid with a carrier such as liposome, high-molecular micelle, orcationic carrier. A nucleic acid carrier such as protamine may be used.An affected part is preferably targeted by an antibody or the like boundto such a carrier. In addition, the retention in the blood can beimproved by binding cholesterol or the like to the nucleic acid.

When a nucleic acid encoding the nucleic acid described above in any of(a) to (c) is contained as the cell growth inhibitor, the nucleic acidinserted in a virus vector such as retrovirus, adenovirus, or Sendaivirus or in a non-virus vector such as liposome may be administered tocells.

<Therapeutic Method>

The method of preventing or treating prostate cancer according to thepresent invention is characterized by that a therapeutically effectiveamount of the cell growth inhibitor of the present invention, i.e., (i)the cell growth inhibitor containing a CTBP1-AS expression inhibitor orfunction inhibitor or (ii) the cell growth inhibitor containing a PSFexpression inhibitor or function inhibitor, is administered.

The term “therapeutically effective amount” as used herein means anamount of an acting substance that ameliorates, to a certain extent, oneor a plurality of symptoms to be treated. In prostate cancer, therefore,it means the amount that can achieve at least one of reduction in tumorsize, inhibition (retardation or termination) of metastasis of tumor,inhibition (retardation or termination) of tumor growth, and relaxationof one or more symptoms related to cancer. The cell growth inhibitor canbe administered as the above-mentioned pharmaceutical composition.

<Screening Method>

In a first mode, a method of screening cell growth inhibitors accordingto the present invention includes a step of bringing cells expressingCTBP1-AS or a cell extract thereof into contact with test compounds inthe presence of androgen, a step of measuring the expression level ofCTBP1-AS, and a step of selecting, from the test compounds, testcompounds decreasing the expression level of CTBP1-AS compared with anexpression level measured in the absence of the test compounds

The cells in which CTBP1-AS has been expressed are not particularlylimited insofar as they are cells in which CTBP1-AS has been expressed.For example, a variety of human cancer cells can be used. Of these,prostate cancer cells (LnCap cells and the like) showing high expressionof CTBP1-AS and metastatic cancer cells thereof are preferred. Uterinecervix cancer cell strain HeLa cells, cell line 293 cells derived fromkidney, mammary tumor cell line MCF27 cells, and the like can also beused. Cells in which CTBP1-AS has not been expressed sufficiently can beused as the cells in which CTBP1-AS has been expressed afterintroduction of an expression vector containing a DNA encoding CTBP1-AS.

In the screening method of the present invention, not cells themselvesbut a cell extract can be used. The cell extract can be prepared in aknown manner by those skilled in the art.

Although test compounds to be used in the screening method of thepresent invention are not particularly limited, examples include organiccompounds, inorganic compounds, nucleic acids (antisense nucleic acids,ribozymes, siRNAs, RNA adaptors, and the like), peptides, proteins(antibodies), and saccharides.

The test compounds can be brought into contact with cells in whichCTBP1-AS has been expressed or a cell extract thereof, for example, byadding the test compounds to a medium of the cells or the cell extract.

When a nucleic acid is used as the test compound, the test compound canbe introduced into cells by using cationic liposome method,electroporation method, microinjection method or the like. When siRNA isused as the test compound, a commercially available transfection kit maybe used according to its instruction.

By bringing the test compounds into contact with cells or a cell extractin the presence of androgen, CTBP1-AS expression can be enhanced stably,making it possible to confirm the effect of the test compounds. Forexample, it is recommended to add androgen to the medium of cells or acell extract three days after bringing the test compounds into contactwith the medium or cell extract.

The step of measuring the expression level of CTBP1-AS can be performedby any method capable of measuring the expression amount of mRNA. Forexample, RNA collected from cells can be analyzed using Real-time PCR ornorthern hybridization method. It is to be noted that CTBP1-AS is anon-coding RNA so that the term “expression level” means an expressionlevel of RNA.

The term “measured in the absence of the test compounds” means that theexpression level of CTBP1-AS is measured by adding only androgen,without adding the test compounds, to similar cells or cell extract tothose or that with which the test compounds are brought into contact.When compared with the value measured in this case, the value issignificantly lower when measured by bringing the test compounds intocontact with the cell or cell extract, the test compounds are selectedas a cell growth inhibitor for cells showing enhanced CTBP1-ASexpression.

In a second mode, a method of screening cell growth inhibitors accordingto the present invention includes a step of bringing cells in which aCTBP1 gene has been expressed into contact with test compounds in thepresence of androgen, a step of measuring the expression level of theCTBP1 gene, and a step of selecting, from the test compounds, testcompounds increasing the expression level of the CTBP1 gene comparedwith an expression level measured in the absence of the test compounds.

From the first mode, the second mode is different in using cells inwhich a CTBP1 gene has been expressed and selecting the test compoundsincreasing the expression level of the CTBP1 gene.

As the cells in which CTBP1 gene has been expressed, for example,prostate cancer cells (LNCap cells, etc.) or metastatic cancer cellsthereof can be used. The cells in which CTBP1 has been expressed canalso be prepared by introducing, into cells in which the CTBP1 gene hasnot been expressed, an expression vector containing a DNA encoding theCTBP1 gene. When the expression vector is introduced, a portion of theCTBP1 gene may be expressed not only by using a DNA encoding the fulllength of the CTBP1 gene but also by using a DNA encoding a portion ofit. The CTBP1 gene encodes a CTBP1 protein so that the term “expression”embraces both transcription and translation.

In the expression level of the CTB1 gene, either the mRNA levelexpression or the protein level expression can be measured by thoseskilled in the art in a known manner. The mRNA level expression can bemeasured using Real-time PCR or northern hybridization similar to themeasurement of CTBP1-AS. The protein level expression can also bemeasured by using electrophoresis such as SDS-PAGE or western blottingusing an antibody against CTBP1 protein.

As described above, inhibition of the expression or function of CTBP1-ASleads to an increase in expression of CTBP1 protein, suppression oftranscriptional activity of AR, and suppression of proliferation ofcells showing enhanced CTBP1-AS expression. It is therefore possible toobtain proliferation inhibitors for cells having enhanced CTBP1-ASexpression by selecting test compounds capable of increasing theexpression level of the CTBP1 gene compared with the expression levelmeasured in the absence of the test compounds.

The screening methods of the present invention may further include astep of administering the test compounds to cells having enhancedCTBP1-AS expression such as prostate cancer cells and performing cellproliferation assay (MTS assay) or a step of administering the testcompounds to cancer-carrying animals to confirm the effect of themagainst the proliferation of cancer cells. By these steps, the effect ofthe test compounds can be examined further.

<Test Method>

The test method for evaluating the prognosis of prostate canceraccording to the present invention includes a step of measuring theexpression level of CTBP1-AS or a CTBP1 gene in cells collected from theprostate gland of a patient and a step of comparing the expression levelwith the expression level in normal prostate cells.

The present inventors have confirmed that the expression amount ofCTBP1-AS in the prostate cancer cells of a patient and metastatic cancercell thereof is significantly greater than that in normal cells and theexpression amount of a CTBP1-gene is significantly smaller than that innormal cells. In addition, they have found that an increase in theexpression amount of CTBP1-AS or a decrease in the expression amount ofthe CTBP1 gene has a correlation with the prognosis of a prostate cancerpatient.

Accordingly, when as a result of the test using the test method of thepresent invention, expression of CTBP1-AS is significantly higher thanthat in normal cells, there is a high possibility of poor prognosis,while when the expression of CTB1 is significantly smaller than that innormal cells, there is a high possibility of good prognosis.

EXAMPLES

The present invention will hereinafter be described in detail based onExamples, but it should be noted that the invention is not limited bythem.

I. The Cell Growth Inhibitor Containing CTBP1-AS Expression Inhibitor orFunction Inhibitor

Methods employed in the following Examples will next be described.

[RNA Fish and Fluorescent Immunostaining]

An antisense CTBP1-AS probe for detecting CTBP1-AS was prepared by usingan RNA labeling kit (Roche) according to its manual. For colocalizationstudy, LNCaP cells were cultured on a cover glass in a 24-well culturevessel. Twenty four hours after treatment with androgen, the cells werewashed with PBS, allowed to stand in 3.6% formaldehyde and 10% aceticacid (in PBS) at room temperature for 20 minutes to fix them, andallowed to stand in 0.5% Triton X-100 at room temperature for 5 minutesfor permeabilization. After washing with PBS, the cells were hybridizedwith the probe (overnight at 42° C. in a moist chamber) and detectedusing a donkey anti-DIG antibody bound to Alexa555. Endogenous PSF wasreacted overnight with a rabbit anti-PSF antibody at 4° C. and detectedusing an anti-rabbit IgG antibody bound to Alexa488, followed by imagingusing a confocal microscope.

[RNA Immunoprecipitation (RIP)]

Confluent LNCaP cells were collected on a 15-cm dish and re-suspended in1 ml NP40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, and 1% NP40). Anantibody to Sin3A, PSF, or NONO or IgG (Santa cruz Biotechnology, Sigma,BD, or Sigma, respectively) was added to the supernatant and theresulting mixture was rotated overnight at 4° C. Protein G beads (30 μL)were added and the mixture was incubated at 4° C. for 2 hours whilemildly rotating it. The beads were washed three times with a lysisbuffer and then re-suspended in 1 ml ISOGEN. The RNA co-precipitated wasisolated and the mRNA level of CTBP1-AS was analyzed using qRT-PCR.

[Northern Blotting]

Preparation of an antisense CTBP1-AS probe and subsequent analysis bynorthern blotting were conducted by using a Northern blot starter kit(Roche) according to its manual. Total RNA (1 μg) was modified and thenloaded on a formaldehyde-containing agarose gel (in 1×MOPS buffer). RNAwas transferred on a Hybond-XL membrane (Roche) and detected using anRNA probe labeled with DIG and extending across the center portion ofCTBP1-AS.

[Microarray]

The total RNA of LNCaP cells was collected using an ISOGEN reagent(Nippon Gene). As an expression analysis microarray, Gene chip humanexon 1.0 ST array (Affymetrix) was used according to its manual. Datawere analyzed using Affymetrix Microarray Suite software. With regard toa comparison array, data from all probe sets were standardized.

[ChIP and Quantitative Real Time PCR]

ChIP was performed in a conventional manner (Takayama K, et al. Oncogene26, 4453-63 (2007)). After treatment for 5 minutes with 1% formaldehydeto crosslink protein and DNA, the cells were collected and a protein-DNAextract was prepared. The extract was subjected to sonication. Afterreaction overnight with a specific antibody and a non-specific IgG, theprotein-DNA complex was collected using a protein A/G agarose. Thecomplex was then washed and reacted overnight at 65° C. to de-crosslinkit. DNA was collected by ethanol precipitation. Fold enrichment relativeto IgG control was quantified by real time PCR using SYBR green PCRmaster mix (Applied Biosystems) and ABI Prism 7000 system (product ofApplied Biosystems) based on SYBR green I fluorescence. PCR productsthus obtained respectively were relatively quantified by the Comparativecycle threshold method (Ct method). GAPDH was used as an externalstandard. The sequences of the primers used are shown in the followingtable.

TABLE 2 Gene Primer SEQ ID NO. CTBP1 F: GGACGCCTGTATGGAAGCA 105(intron 1) R: TCCGCAGACGCCTTTTG 106 CTBP1-ARBSF: GCACTGTGTGGCATAAAAAGAAAA 107 R: TGGAACGTGCCCCAGAA 108[Real Time RT-PCR]

The total RNA was isolated using an ISOGEN reagent. The first cDNAstrand was produced using a Primescript RT reagent kit (Takara) and mRNAwas quantified using real time PCR.

The primers used are shown in the following table.

TABLE 3 Gene Primer SEQ ID NO. CTBP1 F: TGGCCACTGTGGCCTTCT 109R: CGTTCAGGACCTTCTCATGGA 110 CTBP1-AS F: AACCTGGCAGCACGGAAGT 111R: GAGCACAACCACCACCTCATC 112 TMPRSS2 F: TCAACCCCTCTAACTGGTGTGA 113R: AGGCGAACACACCGATTCTC 114 SMAD3 F: CCCCAGAGCAATATTCCAGA 115R: GGCTCGCAGTAGGTAACTGG 116 p53 F: CCCCTCTGAGTCAGGAAACA 117R: TCATCTGGACCTGGGTCTTC 118[siRNA]

A Stealth RNAi system targeting CTBP1-AS, CTBP1 (HSS102437),NONO(HSS143135), PSF (HSS109643), and Sin3A (HSS177954) and a negativecontrol were purchased from Invitrogen. Transfection of cells wasconducted using a Lipofectamine RNAi MAX reagent (Invitrogen) from 48 to72 hours before the test. The sequence of siRNA against CTBP1-AS issiRNA 5′-UUAUGUCUCCAGCAAGCUUGGUCUU.

[In Vivo Tumor Formation Assay]

LNCaP cells (3×10⁶ cells) or LTAD cells (1×10⁷ cells) were transdermallyinjected to both sides of 20 male, 5-week-old nude mice. From the micetransplanted with the LTAD cells, the testicle was removed by surgery atthe time when the tumor volume reached 100 mm³. Three times a week, 5 μgof siRNA against CTBP1-AS or control siRNA was transfected into thetumor by using Lipofectamine RNAi MAX transfection Reagent. The tumorvolume was determined according to the following formula: 0.5×r1×r2×r3(r1<r2<r3).

[Cell Proliferation Assay]

Cells were cultured on a 96 well plate at 3×10³ cells/well. Stable celllines of pcDNA3 CTBP1-AS were inoculated onto a 1%-FBS-containing PRMImedium. For RNAi test, the cells were transfected with stealth RNA 24hours after transfer to the plate. MTS assay was performed using a celltiter reagent (Promega) according to its manual. The test was repeatedfive times for each.

[Cell Culture and Reagent]

LNCaP cells (human prostate cancer cells) were cultured on a RPMI mediumcontaining 10% FBS, 50 units/ml of penicillin, and 50 μg/ml ofstreptomycin. Prior to treatment with androgen, the cells were culturedfor from 48 hours to 72 hours on a phenol red free medium containing 5%dextran charcoal stripped FBS. The antibodies used were Sin3A (AK-11),p53 (Pab-240), p21 (F-5), CyclinD1 (C-20), CyclinA (H-432), CyclinB1(H-433) (Santa cruz Biotechnology), CTBP1 (#612042), NONO (#611278) (BDbioscience), ACH3K9 (#07-352) (Upstate), PSF (#61045) (NovusBiologicals), and SMAD3 (#04-1035) (Millipore). Antibodies against AR,ACH3, and β-actin are described in the report of Takayama, et al.(Takayama K, et al. Oncogene 26, 4453-63 (2007); Cancer Res.69(1):137-42 (2009); Oncogene 30(5):619-30 (2011)). Dihydrotestosteroneand Bicaltamide were purchased from Wako Pure Chemical Industries.

[Western Blotting, Immunoprecipitation]

Conducted in a conventional manner (Takayama K, et al. Oncogene 26,4453-63 (2007)). The cells were collected in an NP40 buffer and aprotein extract was prepared. AN adequate amount of the extract wasboiled for 5 minutes in a Laemmli sample buffer and separated usingSDS-PAGE. After transcription to a PVDF membrane, it was blocked with 5%non-fat dry milk and reacted overnight with a primary antibody at 4° C.The reaction product was then reacted for one hour with a secondaryantibody against rabbit or mouse IgG, followed by color development andphotographing.

[Luciferase Assay]

Prior to transfection, LNCaP cells were incubated for 24 hours on aphenol red free medium containing 5% charcoal stripped FBS. Then, with aFuGENE6 reagent (Roche Diagnostics), the cells were transfected with anARBS-containing pGL3 vector and tk-PRL. Twenty four hours aftertransfection, luciferase activity was measured in a conventional manner.In a luciferase assay using RNAi in combination, the transfection of RNAwas conducted 72 hours before stimulation with androgen.

[Analysis of Cell Cycle]

After transfection of control siRNA or siRNA against PSF, incubation wasconducted for 96 hours and cells were collected. The cells werecentrifuged and washed with PBS. While agitating mildly, 3 ml ofice-cooled 70% ethanol was added slowly to fix them. Until use, theywere stored at 4° C. On the cell cycle analysis day, the cells werecentrifuged, washed with PBS, re-suspended in PBS containing 100 μg/mlRNaseA (Takara) at 10⁶ cells/ml, and incubated at 37° C. for 30 minutes.In order to measure the DNA content, 30,000 cells were analyzed by FACSCalibur flow cytometry using Cell Quest software (BD Biosciences).

[Analysis of Microarray Data]

Microarray analyses of siRNA against CTBP1-AS and siRNA against PSF wereconducted, independently. As a result of a test using siRNA against PSF,700 genes are presumed to be suppressed by PSF. The function of thesetarget genes were identified using DAVID and for pathway analysis,REACTOME was used. In each analysis, 300 genes obtained from the controlsample were identified as genes suppressed by androgen.

<Discovery of CTBP1-AS>

Considering that inhibition of signals downstream of AR is effective asa target of prostate cancer treatment, the present inventors haveestablished a method of finding a gene directly targeted by AR by usingchromatin immunoprecipitation and tiling array in combination (ChIP-chipmethod) (Takayama K. et al. Oncogene 26, 2007, 4453-4463, Takayama K. etal. Cancer Res. 69, 2009, 137-142). Moreover, to elucidate thetranscription network by androgen, ChIP-chip analysis was conducted onthe whole human genome level by using, in addition to AR, an antibodyagainst acetylation at histone H3K9/K14 to identify the AR binding site(ARBS) and the histone H3 acetylation site (ACH3).

In addition, Cap Analysis gene expression (CAGE) was conducted usingLNCaP cells. CAGE is a method of sequencing concatemers of DNA tagsderived from 20 bases from the 5′ end of mRNA, carrying out genome-wideidentification of a starting point of transcription, and profiling thepromoter usage and is therefore a high throughput method. A region whereCAGE tags were mapped to the human genome and aggregated as a result ofCAGE analysis was defined as a tag cluster (TC). Of the TCs, thoseundergoing a significant change in the distribution of tags wereidentified.

Based on the recent report (Katayama S. et al. Science 309, 2005,1564-1566) that an antisense transcription product controls theexpression of genes in the vicinity of the product and the finding thattranscription control of AR is an important signal of the onset ofprostate cancer, the relationship between the transcriptional activityof AR and an antisense transcription product was considered to be animportant factor for describing the advance of cancer. As a result ofsearching for an androgen responsive gene controlled by an antisense,CTBP1 was found.

In addition, in the vicinity of ARBS of CTBP1, an antisense TC to beactivated by androgen was found. According to GENBANK, it was found tohave ex3 of AX747592 as a starting point of transcription. The antisenseRNA was therefore named CTBP1-ASd (SEQ ID NO:20).

CTBP1-ASd contains a sequence encoding the sequence of 330 amino acids.It has been found from the analysis by the present inventors by usingPfam that this amino acid sequence does not encode a protein motif.Analysis using Netstart has revealed that the first methionine isunlikely to function as a starting point of protein translation.Furthermore, as a result of a test by the IVT method (in vitrotranscription method), protein synthesis from CTBP1-ASd was notrecognized (data not shown). These results mean that CTBP1-ASd is anon-coding RNA not encoding a protein motif.

<Preparation of siRNA Having RNAi Effect Against CTBP1-ASd>

As siRNA, stealth RNAi (Invitrogen) designed based on the base sequenceof CTBP1-Asd was used. It is a blunt end type double-stranded RNA havinga length of 25 bases longer than the typical siRNA and is characterizedby that it can minimize the activation of an interferon responsive gene,can prevent an off-target effect derived from the sense strand, and hasvery high stability in the serum. It also has actual using results invivo.

The optimum site of the siRNA sequence was determined using the RNAiDesigner on Invitrogen's website. The sequence of siRNA thus prepared isas described below.

TABLE 4 Abbreviation Target siRNA siRNA[1] 1804-1828UGACCAGUCCGUUUGACACUGAGUG (SEQ ID NO. 7)CACUCAGUGUCAAACGGACUGGUCA (SEQ ID NO. 8) siRNA[2] 1894-1918UUGAGAUGCCGGAAACAUUGAUGGG (SEQ ID NO. 9)CCCAUCAAUGUUUCCGGCAUCUCAA (SEQ ID NO. 10) siRNA[3] 2348-2372UUAUGUCUCCAGCAAGCUUGGUCUU (SEQ ID NO. 11)AAGACCAAGCUUGCUGGAGACAUAA (SEQ ID NO. 12) siRNA[4] 3063-3087GAGACAGGAGGAUGUAGUUUCUAAU (SEQ ID NO. 13)AUUAGAAACUACAUCCUCCUGUCUC (SEQ ID NO. 14)

In Examples described below, the above-mentioned siRNA [3] wasadministered to a siRNA administration group. As a control in the siRNAtest (except in vivo test), a double-stranded RNA (Stealth™ RNAiNegative Control Medium GC Double-stranded, Invitrogen 12935-300) notshowing a RNAi effect against CTBP1-ASd was administered.

<Androgen-Dependent Expression of CTBP1-AS and Effect of SiRNAAdministration on Androgen-Dependent Expression Suppression of CTBP1Gene>

In the test, LNCaP cells of an AR-positive human prostate cell line wereused. For cell culture, RPMI1640 (Sigma) containing 10% fatal bovineserum (FBS, Sigma), 100 μg/ml streptomycin, and 100 U/ml penicillin(Invitrogen) was used as a cell broth. The cells were cultured at 37° C.in an incubator containing 5% carbon dioxide gas in the air.

On a 6-well plate, 1×10⁵ LNCaP cells were cultured. On the followingday, 20 nM and 50 nM siRNAs were introduced into the cells by usingHiperfect Transfection Reagent (Quiagen) and OPTI-MEM (Invitrogen).Forty eight hours later, the resulting cells were stimulated with 10 nMandrogen R1881 or Vehicle. After culturing for further 24 hours, RNA wascollected using ISOGEN (Nippon gene).

Expression analysis of mRNA was conducted using Real-time PCR (Prism7000system; Applied biosystems). From an amplification curve available bythe Real-time PCR method, an intracellular expression amount of atranscription product was determined by the ΔΔCt method. The expressionamount was corrected based on the expression amount of a GADPH gene.

Measurement results of the CTBP1-AS expression are shown in FIG. 1. InControl, expression of CTBP1-AS was accelerated androgen-dependently,but in the cells of the siRNA administration group, CTBP1-AS expressionwas suppressed in a siRNA-concentration dependent manner. In the cellsadministered with 50 nM siRNA, expression induction by androgen wassuppressed by from 60 to 80%.

The measurement results of the expression of mRNA of CTBP1 are shown inFIG. 2. In Control, the expression of mRNA of CTBP1 was suppressed byandrogen. On the other hand, in the siRNA administration group, theexpression suppression effect of CTBP1 by androgen disappeared in asiRNA-concentration dependent manner.

In addition, 24 hours after treatment with 10 nM R1881, a protein wascollected and the protein-level expression amount of CTBP1 was measuredby western blotting. The results are shown in FIG. 3. The protein-levelexpression was also suppressed by androgen administration, but due tothe administration of siRNA, the expression suppression effect decreasedin a siRNA-concentration dependent manner.

<Luciferase Assay>

Luciferase assay was conducted to confirm the influence of CTBP1-AS ontranscriptional activity of AR.

A luciferase vector was prepared by inserting, in the promoter region ofpGL3-basic (Promega), the promoter and enhancer of PSA (Prostatespecific antigen), that is, a typical androgen responsive gene(PSA-LUC). PSA-LUC has an AR binding site and the promoter is activatedandrogen-responsively.

LNCaP cells were sprayed and cultured on a 24-well plate at 3×10⁴cells/well. After culturing for 2 days on a phenol red-free mediumcontaining 2.5% charcoal serum, PSA-LUC was introduced. On the followingday, the resulting cells were stimulated with 10 nM R1881 or Vehicle.The cells were collected after 24 hours. Luciferase activity wasmeasured using a Dual-Luciferase Reporter Assay System (Promega).

Two days before introduction of the vector into the cells, siRNA andcontrol double-stranded RNA were administered by the above-describedmethod.

The results are shown in FIG. 4A. It has been demonstrated in Controlthat luciferase activity showed a marked increase and administration ofandrogen enhanced AR transcriptional activity, which activated the PSApromoter and enhancer. In the siRNA administration group, on the otherhand, luciferase activity was markedly suppressed.

The results of studying the expression of an androgen responsive gene inthe same cells are shown in FIGS. 4B and 4C. In Control, the expressionamounts of FKBP5 and TMPRSS2, that is, typical androgen responsivegenes, increased, while in the siRNA administration group, an increasein the expression amount was suppressed in a concentration dependentmanner.

It has been confirmed from the above-mentioned results that suppressionof CTBP1-AS leads to suppression of AR transcriptional activity.

<Influence of Function Suppression of CTBP1-AS on Proliferation of LNCaPCells>

The cell-level influence of CTBP1-AS on LNCaP cell was analyzed usingMTS assay.

On a 96-well plate, LNCaP cells were continuously cultured at 3×10³cells/well. Similar to luciferase assay, siRNA was administered on thefollowing day. Forty eight hours, 72 hours, and 96 hours afteradministration, the resulting cells were reacted for one hour by usingCell titer 96 (Promega).

MTS assay is a method of measuring a viable cell count based on areduction reaction for converting, via PES (phenazine ethosulfate), atetrazolium salt(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS) into a formazan product which is a chromogenicsubstance. The cell proliferation ability was measured at an absorbanceof 490 nm by using a microplate reader.

The results are shown in FIG. 5. It has been found that in the siRNAadministration group, compared with Control, cell proliferation wassignificantly suppressed and CTBP1-AS promoted the proliferation ofLNCaP cells.

<Influence of Function Suppression of CTBP1-AS on Tumor Proliferation InVivo>

Influence of CTBP1-AS and function suppression of CTBP1-AS on the tumorproliferation in vivo was analyzed.

To subcutaneously transplant LNCaP cells to mice, they were mixed withPBS and matrigel (BD bioscience). The mixture for 20 mice was preparedat a time so that the cells, PBS, and matrigel per mouse be 1×10⁷cells/mice, 100 μl, and 100 μl, respectively.

The mixture was injected subcutaneously to 6-week-old male BALB/c nudemice (CLEA Japan) by using a 25G injection needle.

Injection of siRNA into tumor was conducted using Gene silencer(Genlantis). A mixture obtained by mixing 5 μg of siRNA with 5 μl ofGene silencer in OPTI-MEM was locally injected into the mouse tumor. Incontrol group, a double-stranded RNA having the following base sequencewas similarly administered.

(SEQ ID NO: 15) AAGACGAUCGUUCGGGACAACAUAA (SEQ ID NO: 16)UUAUGUUGUCCCGAACGAUCGUCUU

The siRNA injection was started two weeks after the cancer cellstransplanted into mice seemed to be subcutaneously engrafted and it wasconducted twice a week.

The size of the mouse tumor was measured once a week. The major axis(r1) and the minor axes (r2, r3) were measured at two positions and thesize of the tumor was determined based on the formula: (r1×r2×r3)/2.

Ten weeks after transplantation, mice were sacrificed and their tumorwas excised from under the skin. After the tumor was weighed, a portionof it was provided for RNA extraction using ISOGEN. After cDNAsynthesis, RNA level expression amounts of CTBP1-AS and CTBP1 weremeasured using Real time-PCR. Another portion was dissolved in an SDSlysis buffer (10 mM Tris-HCl, pH7.5, 2% SDS, 10% mercaptoethanol). Theresulting solution was provided for protein extraction and a CTBP1expression amount was measured using western blotting.

The results are shown in from FIG. 6 to FIG. 9.

As shown in FIG. 6, in the siRNA administration group, compared withControl, proliferation of the tumor was significantly suppressed(p<0.01). As shown in FIG. 7, it has been observed that in the siRNAadministration group, an increase in the tumor weight was suppressedmarkedly (p<0.001).

FIG. 8 shows the measurement results of the RNA level expression amountof CTBP1-AS and CTBP1 in the tumor by using Real time-PCR. Compared withControl group, the expression level of CTBP1-AS in the tumor of fourmice was reduced by administration of RNAi against CTBP1-AS. On theother hand, the expression amount of CTBP1 was increased by theadministration of RNAi against CTBP1-AS compared with Control group.These results have revealed that also in vivo, CTBP1 is under negativecontrol of CTBP1-AS.

FIG. 9 shows the measurement results of the protein-level expressionamount of CTBP1 in tumor by western blotting. In the RNAi administrationgroup, compared with Control group, the protein level expression amountof CTBP1 was also increased.

It has been confirmed from the above results that a mechanism ofsuppressing CTBP1 by CTBP1-AS also functions in in vivo tumorproliferation in the tumor model and suppression of tumor proliferationis promoted by the suppression targeting CTBP1-AS.

<Expression of CTBP1-AS in Actual Tumor>

Expression profile data (GSE3325) of prostate and prostate cancer casesusing Human Genome U133 Plus 2.0 array of Affymetrix made public in GEODatasets on NCBI (http://www.ncbi.nlm.nih.gov) were downloaded.

Signal intensity was extracted while paying attention to the probe(1563571_at) specific to CTBP1-AS (AX747592) of normal, cancer, andmetastasis samples. Mann-Whitney U test was conducted and the expressionamount was compared among these groups.

The results are shown in FIG. 10. The expression amount of CTBP1-AS incancer and the cancer tissue of distant metastasis were significantlyhigher than that of normal prostate (p<0.05, Mann-Whitney U test). Ithas been presumed from this result that CTBP1-AS is involved in theonset and development of cancer.

<Expression of CTBP1 in Actual Cancer>

Expression of CTBP1 in the actual prostate cancer was assessed byimmunostaining. Immunostaining was performed according to the method ofSuzuki, et al. (Suzuki T. et al. Clin. Cancer Res. 2005 Sep. 1; 11(17):6148-54).

Cases were selected from 104 cases with a surgical specimen of prostatecancer excised from 1987 to 2001 in the Urology/the University of TokyoHospital. After 99 sections corresponding to the normal site and 101sections corresponding to the prostate cancer were fixed in formalin,paraffin sections were prepared. As a primary antibody, CTBP1-specificmouse monoclonal antibody (BD Bioscience) was used. With Histofine Kit(Nichirei) for immunohistochemical staining, staining was conducted witha 3,3′-diaminobenzidine solution [1 mmol/L 3,3′-diaminobenzidine, 50mmol/L Tris-HCl buffer (PH7.6), and 0.006% H₂O₂]. Images of sectionsrepresentative of normal prostate and prostate cancer are shown in FIG.11C.

A labeling index (LI) in the nucleus of CTBP1 in each section wasmeasured. The expression of CTBP1 protein in the normal tissue andcancer tissue was quantified and compared.

The results are show in FIG. 11A. The expression amount of CTBP1decreased in prostate cancer cells compared with that in normal cells.

With regards to 101 prostate cancer cases, a survival rate was comparedbetween a CTBP1 high expression group and a CTBP1 low expression groupbased on the data obtained by tracking the progress on an outpatientbasis every three months after surgery for five years and the resultswere analyzed using the Kaplan-Meier method with the Long-rank test. Theclassification into the high expression group and the low expressiongroup was conducted based on the median of Labeling Indices showing theexpression of CTBP1.

The results are shown in FIG. 11B. The survival rate was significantlyhigh in the CTBP1 high expression group compared with that in the lowexpression group.

It has been confirmed from the above results that a suppressingmechanism of CTBP1 by CTBP1-AS also functions in proliferation of actualtumor.

<Identification of CTBP1-AS Except CTBP1-ASd>

CTBP1-ASs except CTBP1-ASd expressed in prostate cancer cells weresearched in the following manner.

Twenty four hours after stimulation of LNCaP cells with 10 nM R1881, RNAwas collected. RNA (1 μg) was treated with 1 μl of DNase (Roche, 10U/μl) and a reverse transcription reaction was then conducted using aRACE Kit (Roche). Next, a PCR reaction was conducted using primersobtained by the CAGE method, one of which was in the vicinity of astarting point of transcription and the other one had anadapter-specific sequence at the terminal. The PCR product thus obtainedwas inserted into a plasmid by using a TA Cloning Kit (Invitrogen). Theplasmid was sequenced and the sequence of the transcription productobtained by the PCR reaction was analyzed. The sequence thus obtainedwas mapped to a human genome sequence and a range of the transcriptionproduct was determined.

The following are the 3′ RACE primer and PCR adaptor primer used.

3′RACE primer: (SEQ ID NO: 21)5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3′ PCR adaptor primer:(SEQ ID NO: 22) 5′-GACCACGCGTATCGATGTCGAC-3′

The results show that there are at least four kinds of CTBP1-AS as shownin FIG. 12. One of them is the above-described CTBP1-ASd. The basesequence of CTBP1-ASa is shown in SEQ ID NO:19, the base sequence ofCTBP1-ASb is shown in SEQ ID NO:18, and the base sequence of CTBP1-AScis shown in SEQ ID NO:17.

These CTBP1-Ass each contain the target sequence of siRNA described inthe above table. It is therefore presumed that in the expressionsuppression test using siRNA in the above Example, expression of theseCTBP1-AS was also suppressed.

A probe for detecting, as a sequence which CTBP1-ASa to CTBP1-ASd havein common, a region (SEQ ID NO:23) from position 1286 to position 3515of the RNA sequence described in SEQ ID NO:1 was prepared and northernblotting was conducted. The probe was prepared following the protocol byusing DIG Northern Starter Kit (Roche).

LNCaP cells were stimulated with 10 nM R1881 and 0, 6, 12, 18, 24, and48 hours later, RNAs were collected. RNAs (each, 1 μg) wereelectrophoresed on an agarose gel containing formaldehyde andtransferred to a membrane. The membrane was hybridized with the aboveprobe (100 ng/ml) and a DIG-specific antibody was used for detection.

In order to measure the amount of RNA used in the test, a probe (Roche)against β actin was purchased at the same time and the same membrane wasused for detection.

The results are shown in FIG. 13. A transcription product having alength of about 5 kb was detected, suggesting that among theabove-mentioned four kinds of CTBP1-ASs, CTBP1-ASb was expressed mainly.

<Localization of CTBP1-AS>

LNCaP cells were treated for 24 hours with 10 nM R1881 or Vehicle. Fromthe cells collected, intranuclear RNA and cytoplasmic RNA were extractedusing a PARIS kit (Ambion). From respective samples of them, 1 μg of RNAwas electrophoresed and an expression amount was analyzed using northernblotting. As Loading Control, a probe against β-actin was hybridized andan expression amount was measured. The expression amount of each of thegenes was corrected by GAPDH.

The results are shown in FIG. 14. Intranuclear localization of CTBP1-ASwas confirmed.

<Involvement of CTBP1-AS in Androgen-Depletion Resistant Cancer CellProliferation—In Vitro>

Lysates of LTAD cells and LNCaP cells (2.5% charcoal serum, cultured for3 days, stimulated with R1881, R1881+Bicaltamide added) and westernblotting was conducted using a CTBP1 antibody. A probe against β-actinwas used as a Roding Control.

In order to compare the expression amount of CTBP1-AS between LTAD cellsand LNCaP cells, Total RNA of LTAD cells and LNCaP cells (2.5% charcoalserum, cultured for 3 days, stimulated with R1881, R1881+Bicaltamideadded) were collected. The RNA (1 μg) was electrophoresed, transferred,and subjected to northern blotting with a probe against CTBP1-AS.

The results are shown in FIGS. 15 a and 15 b. The LTAD cells werecultured under an androgen-depleted state to be a hormone therapyresistant model. When the LNCaP cells were stimulated with androgen,expression of CTBP1 was decreased to a similar level to that of the LTADcells. In the LTAD cells, expression of CTBP1-AS was increasedremarkably.

Next, an influence of CTBP1-AS on hormone-dependent or hormone-depletedproliferation of prostate cancer cells was analyzed.

LNCaP cells were sprayed on a phenol red-free medium containing 2.5%charcoal serum at 3×10³ cells/well. On the following day, RNAi againstCTBP1-AS was transfected. Two days later, the cells were stimulated withEt or R1881 and on Day 1, 3, and 5, MTS assay was performed to evaluateproliferation ability.

Separately, the LTAD cells were sprayed on a phenol red-free mediumcontaining 2.5% charcoal serum at 3×10³ cells/well. On the followingday, RNAi against CTBP1-AS was transfected. Two days later, the cellswere stimulated with Et or R1881 and on Day 1, 3, and 5, MTS assay wasperformed to evaluate proliferation ability.

The results are shown in FIG. 15 c. Cancer cells proliferate evenwithout stimulating the androgen depletion resistant LTAD cells withandrogen. Either androgen-dependent proliferation orandrogen-independent proliferation was suppressed by knockdown ofCTBP1-AS.

<Involvement of CTBP1-AS in Androgen-Depletion Resistant Cancer CellProliferation—In Vitro>

The role of CTBP1-AS in proliferation of hormone therapy resistant cellswas analyzed in vivo.

LTAD cells were subcutaneously transplanted to nude mice at 1×10⁷cells/mouse. The testicle was removed by surgery at the time when thetumor volume reached 100 mm³. Control siRNA or siRNA against CTBP1-ASwas locally injected (5 μg/mouse, twice/week).

The results are shown in FIG. 16. Even in vivo, by suppressingexpression of CTBP1-AS, an increase in the volume of theandrogen-depletion resistant tumor was suppressed (FIGS. 16 a and 16 b).As a result of measurement of the expression amount of CTBP1-AS in tumorby qRT-PCR, decrease in the mRNA level expression has been confirmed(FIG. 16 c). On the other hand, CTBP1 in tumor was measured by westernblotting. Due to knock down of CTBP1-AS, the expression amount of CTBP1increased (FIG. 16 d).

<Involvement of RNA-Binding Protein PSF in Expression Suppression ofCTBP1>

Expression of three genes, that is, PSF, NONO, and Sin3A was suppressedby RNAi. LNCaP cells were transfected with siRNAs against PSF, NONO, andSin3A. Two days later, lysates were collected and the expression amountwas examined by western blotting. It has been confirmed that any of thesiRNAs specifically suppressed the target expression.

LNCaP cells were treated with ethanol or R1881 for 24 hours and two daysbefore stimulation, the resulting cells were transfected with controlsiRNA or siRNA against PSF. The RNA was collected, cDNA was synthesized,and an expression amount of CTBP1 was analyzed by RT-PCR.

The results are shown in FIG. 17 b. When the expression of PSF wassuppressed, the suppression degree of CTBP1 expression by stimulationwith androgen decreased.

In addition, ChIP was conducted using an ACH3 antibody. A histoneacetylation level of a promoter region of CTBP1 and CTBP1-AS wasmeasured using the real time PCR, followed by correction with GAPDH. Themyoglobin exon region was used as a negative control.

The results are shown in FIG. 17 c. When the expression of PSF wassuppressed, the suppression degree of CTBP1 expression by stimulationwith androgen decreased. This has suggested a promoter-level involvementof PSF in CTBP1 expression.

<Androgen-Dependent Binding of PSF to CTBP1-AS>

PSF is a RNA-binding transcriptional repressor. The presence or absenceof binding between PSF and CTBP1-AS was analyzed.

LNCaP cells were treated with ethanol or R1881 for 24 24 hours and twodays before stimulation, the resulting cells were transfected withcontrol siRNA or siRNA against PSF. After a nuclear compartment wasextracted and a lysate was collected, immunoprecipitation was conductedusing an antibody specific to Norman IgG, PSF and NONO.Immunoprecipitation was conducted using Protein G and after washing, RNAwas extracted using Isogen. A reverse transcription reaction wasperformed using total RNA and expression amounts of GAPDH, Myoglobin(MG), and CTBP1-AS were measured using the real time RT-PCR.

The results are shown in FIG. 18. It has been confirmed that CTBP1-ASbinds to PSF in an androgen-dependent manner.

<Analysis of CTBP1-AS Localization by RNA-FISH>

Intranuclear localization of PSF and CTBP1-AS was analyzed. Ethanol orR1881 was added to LNCaP cells, followed by reaction for 24 hours. Then,RNA FISH of CTBP1-AS was performed. After fixed with formalin andmethanol, the cells were reacted overnight at 42° C. for binding byusing a probe against DIG-labeled CTBP1-AS. On the following day, afluorescence-labeled antibody against DIG was bound and after washing,immunostaining with an anti-PSF antibody was performed. A secondantibody against a mouse antibody was reacted for detection. The nucleuswas stained with DAPI.

The results are shown in FIG. 19. PSF and CTBP1-AS showed almost thesame localization, suggesting that they have been bound to each other.

<Control of Cell Cycle by PSF>

Next, another gene whose transcription was suppressed by PSF wassearched.

Cells were transfected with control siRNA or siRNA against PSF. Twentyfour hours after stimulation with ethanol or R1881, RNA was collected.The RNA was amplified and gene expression was analyzed using amicroarray. From an expression-suppressed gene group and anexpression-enhanced gene group, a gene whose change was suppressed byandrogen was extracted. A change rate of gene expression caused byandrogen was Log 2 transformed and a Heat map was created using Clusterand Tree view (FIG. 20 a). Suppressed transcription is shown with alighter color.

70% of the gene suppressed by androgen was released from the suppressionby siPSF. Moreover, induction of most of the androgen-mediated genes wassuppressed by siPSF. This suggests that PSF widely mediates theexpression suppression by androgen.

The gene group in which PSF was engaged in androgen action was subjectedto pathway analysis. It has been found as a result of analysis of thegene group whose change was suppressed by PSF suppression, among thegene groups suppressed by androgen, that genes involved in cell cyclesuch as p53 and SMAD3 were concentrated (in the drawing, UGT2B15 is anegative control whose expression is suppressed by androgen but is notinfluenced by PSF).

Next, an influence of suppression by PSF on the cell cycle was analyzedusing FACS. LNCaP cells, LTAD cells, and BicR cells were transfectedwith control siRNA or siRNA against PSF and 96 hours later, the cellswere collected. The cell cycle was analyzed using FACS. A change in thepercentage of S-phase cell fraction is shown in FIG. 20 b. When PSF wasknocked down, the tendency to decrease in the percentage of the DNAsynthesis S-phase and retardation of the cell cycle were found.

A similar test to that in FIG. 20 a was conducted except that CTBP1-AS,instead of PSF, was knocked down and kinds of genes to be released fromthe androgen-mediated suppression were compared. The results are shownin FIG. 20 c. About 50% of the cells to be released from theandrogen-mediated suppression by knockdown of PSF showed a tendency ofbeing released from the androgen-mediated suppression even by knockdownof CTBP1-AS. This suggests that CTBP1-AS and PSF are cooperativelyinvolved in androgen-mediated suppression of gene expression.

<Analysis of p53 and SMAD3, Target Genes of PSF>

Influence of PSF on the expression of p53 and SMAD3 was analyzed.

LNCaP cells were transfected with control siRNA or siRNA against PSF and48 hours after the transfection, they were stimulated with ethanol orR1881. Twenty four hours later, RNA and protein were collected.Measurement results of the expression amount of p53 and SMAD3 by qRT-PCRare shown in FIG. 21 a (corrected by GAPDH). The results of westernblotting are shown in FIG. 21 b (β actin was used as Loading control).

Stimulation with androgen decreased both the mRNA level expression andprotein level expression of SMAD3 and p53. When expression of PSF wassuppressed, the androgen-mediated suppression of expression tended to bereleased. This suggests that SMAD3 or p53 is hardly influenced byandrogen without PSF, in other words, suppression of SMAD3 or p53expression by androgen is mediated by PSF.

<Androgen-Dependent Expression Suppression of Cell-Cycle-Related Factorsby CTBP1-AS>

The influence of CTBP1-AS on the expression of p53 and SMAD3 wasanalyzed.

LNCaP cells were transfected with control siRNA or siRNA againstCTBP1-AS and 48 hours later, they were stimulated with ethanol orandrogen. Forty eight hours later, RNA and protein were collected andthe expression amounts of p53, p21, and SMAD3 were analyzed by westernblotting. The results are shown in FIG. 22 a.

Expression of each of p53, p21, and SMAD3 was decreased by stimulationwith androgen, but a decreasing degree was reduced by suppressing theexpression of CTBP1-AS.

LTAD cells, that is, hormone-depletion resistant prostate cancer cellswere transplanted to nude mice. After formation of a tumor, their testiswas excised and locally injected with control siRNA or siRNA againstCTBP1-AS. Three weeks later, the tumor was excised, protein wascollected, and the expression amount of a cell cycle-related geneproduct was analyzed by western blotting. The results are shown in FIG.22 b (each, n=2). Suppression of the expression of CTBP1-AS led torelease of androgen-dependent expression suppression of the cellcycle-related gene product.

FIG. 23 is a schematic view showing a gene expression control mechanismby CTBP1-AS and PSF which is suggested by the above results.

First, when androgen binds to an androgen receptor AR, transcription ofCTBP1-AS increases.

As a result, in a global genome region (within the left framework in thedrawing), CTBP1-AS binds to PSF to form a complex and this complexsuppresses expression of SMAD3 or p53. SMAD3 or p53 is a cell cycleinhibiting gene so that when its expression is suppressed, cellproliferation is promoted.

On the other hand, locally (within the right framework in the drawing),when transcription of CTBP1-AS increases, a complex of CTBP1-AS and PSFbinds to a histone deacetylation enzyme (HDAC) and suppressestranscription of CTBP1. Since CTBP1 is a transcription suppressor of anandrogen receptor, suppression of the transcription of CTBP1 increasesthe transcription of the androgen receptor and activates the receptor.

Thus, either globally or locally, an increase in the transcription ofCTBP1-AS serves to cause proliferation of cancer cells. This mechanismhas proved that suppression of CTBP1-AS or PSF leads to suppression ofthe proliferation of cancer cells.

<Expression Suppression of CTBP1-AS by siRNA>

After designing and synthesizing siRNAs against CTBP1-AS shown in thefollowing table, their effect on the expression suppression of CTBP1-ASwas measured.

TABLE 5 Target sequence SEQ SEQ SEQ Oligo (target sequence for ID ID IDname designing siRNA) NO. >sense_strand NO. >antisense_strand NO. No. 1accaacaggaaacgtcctactta 24 CAACAGGAAACGUCCUACUUA 53AGUAGGACGUUUCCUGUUGGU  79 No. 2 ctcaacatcagaccaattaatta 25CAACAUCAGACCAAUUAAUUA 54 AUUAAUUGGUCUGAUGUUGAG  80 No. 3accaactgtcaagaaacaattag 26 CAACUGUCAAGAAACAAUUAG 55AAUUGUUUCUUGACAGUUGGU  81 No. 4 ctcactgctgctgatgattgtag 27CACUGCUGCUGAUGAUUGUAG 56 ACAAUCAUCAGCAGCAGUGAG  82 No. 5gacataccacacaaacactgatc 28 CAUACCACACAAACACUGAUC 57UCAGUGUUUGUGUGGUAUGUC  83 No. 6 gaccaattaattagaccacaaaa 29CCAAUUAAUUAGACCACAAAA 58 UUGUGGUCUAAUUAAUUGGUC  84 No. 7tcccacatcaacacggtaacacc 30 CCACAUCAACACGGUAACACC 59UGUUACCGUGUUGAUGUGGGA  85 No. 8 cacccggcctaacatggagaatt 31CCCGGCCUAACAUGGAGAAUU 60 UUCUCCAUGUUAGGCCGGGUG  86 No. 9acccggcctaacatggagaattt 32 CCGGCCUAACAUGGAGAAUUU 61AUUCUCCAUGUUAGGCCGGGU  87 No. 10 ggcctcataacgcactaggataa 33CCUGAUAACGCACUAGGAUAA 62 AUCCUAGUGCGUUAUGAGGCC  88 No. 11aacgctgtgggatatgaggttgg 34 CGCUGUGGGAUAUGAGGUUGG 63AACCUCAUAUCCCACAGCGUU  89 No. 12 cccggcctaacatggagaattta 35CGGCCUAACAUGGAGAAUUUA 64 AAUUCUCCAUGUUAGGCCGGG  90 No. 13gcctaacatggagaatttagaac 36 CUAACAUGGAGAAUUUAGAAC 65UCUAAAUUCUCCAUGUUAGGC  91 No. 14 tcctacttaatggtgaaatgtca 37CUACUUAAUGGUGAAAUGUCA 66 ACAUUUCACCAUUAAGUAGGA  92 No. 15ctctatactattaataacagaca 38 CUAUACUAUUAAUAACAGACA 67UCUGUUAUUAAUAGUAUAGAG  93 No. 16 gcctcataacgcactaggataac 39CUCAUAACGCACUAGGAUAAC 68 UAUCCUAGUGCGUUAUGAGGC  94 No. 17ttctcccacatcaacacggtaac 40 CUCCCACAUCAACACGGUAAC 69UACCGUGUUGAUGUGGGAGAA  95 No. 18 gcctcggcctcataacgcactag 41CUCGGCCUCAUAACGCACUAG 70 AGUGCGUUAUGAGGCCGAGGC  96 No. 19tgctgctgatgattgtagctaat 42 CUGCUGAUGAUUGUAGCUAAU 71UAGCUACAAUCAUCAGCAGCA  97 No. 20 aggaactaactctatactattaa 43GAACUAACUCUAUACUAUUAA 72 AAUAGUAUAGAGUUAGUUCCU  98 No. 21cactaagcagagtataggttaaa 44 CUAAGCAGAGUAUAGGUUAAA 73UAACCUAUACUCUGCUUAGUG  99 No. 22 ttaacacctgattaatataaaag 45GACACCUGAUUAAUAUAAAAG 74 UUUAUAUUAAUCAGGUGUCAA 100(ttgacacctgattaatataaaag) (46) No. 23 tgaacactcaagaggcaaagact 47GACACUCAAGAGGCAAAGACU 75 UCUUUGCCUCUUGAGUGUCCA 101(tggacactcaagaggcaaagact) (48) No. 24 gaccgtctaaacgcactacatcc 49CCGUCUAAACGCACUACAUCC 76 AUGUAGUGCGUUUAGACGGUC 102 No. 25cccgcgaacgtggtgtctcctgc 50 CGCGAACGUGGUGUCUCCUGC 77AGGAGACACCACGUUCGCGGG 103 No. 26 tgacaacaagtgtggatgtaaac 51GCAACAAGUGUGGAUGUAAAC 78 UUACAUCCACACUUGUUGCCA 104(tggcaacaagtgtggatgtaaac) (52)

The results are shown in FIG. 24. In this drawing, NC and NC2 arenegative controls. It has been confirmed that compared with the negativecontrols, siRNAs of Nos. 3, 4, 6, 10, 12, 18, and 24 suppress thetranscription of CTBP1-AS.

II. The Cell Growth Inhibitor Containing PSF Expression Inhibitor orFunction Inhibitor

Cell Line

Human prostate cancer cell line LNCaP was grown in RPMI-1640supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/mlstreptomycin. Cells were incubated at 37 deg C. in an atmosphereincluding 5% CO₂.

Design of siRNA

siRNAs targeting PSF was purchased from SIGMA (St. Louis, Mo.). ThesesiRNAs were designed by the inventors' program so that the senseoligonucleotides would not induce off-target effect. SiRNA sequencesdesigned by this program have previously been used successfully for thebiological experiments in vivo. We determined that the siRNA sequencesagainst PSF by surveying all other annotated genes so that the siRNAsequences would not match the gene sequences and confirmed that thepossibility of off-target effect was low. The negative control siRNA(siControl) did not have off-target effects on other mammalian genes.The knockdown effects were evaluated using prostate cancer cells and thesiRNA sequence showing most efficient RNAi effect was selected for thexenograft experiment.

The siRNAs used for the following Examples are listed below.

siPSF Sense strand Antisense strand # (5′->3′) 21mer (5′->3′) 21merTarget sequence 1 GCACGUUUGAGUACGAAUAUU UAUUCGUACUCAAACGUGCCA 1542-1564*CDS** (SEQ ID NO: 119) (SEQ ID NO: 127) 2 GGCACGUUUGAGUACGAAUAUAUUCGUACUCAAACGUGCCAU 1541-1563 CDS (SEQ ID NO: 120) (SEQ ID NO: 128) 3GCAUAUUAGGCUACGUAUUCC AAUACGUAGCCUAAUAUGCAU 2586-2608 3′UTR(SEQ ID NO: 121) (SEQ ID NO: 129) 4 GAUGAUCGUGGAAGAUCUACAUAGAUCUUCCACGAUCAUCCA 1304-1326 CDS (SEQ ID NO: 122) (SEQ ID NO: 130) 5GGGAGAUCCCUAUGGUUCAGG UGAACCAUAGGGAUCUCCCAU 1951-1973 CDS(SEQ ID NO: 123) (SEQ ID NO: 131) 6 GUUUGGGCAGGUAAAAUUAUGUAAUUUUACCUGCCCAAACAG 2355-2377 3′UTR (SEQ ID NO: 124) (SEQ ID NO: 132)7 CAUAGGUUAUGAAGCUAAUCC AUUAGCUUCAUAACCUAUGCC 2008-2030 CDS(SEQ ID NO: 125) (SEQ ID NO: 133) 8 GGCAUAGGUUAUGAAGCUAAUUAGCUUCAUAACCUAUGCCAC 2006-2028 CDS (SEQ ID NO: 126) (SEQ ID NO: 134)*base number **CDS = coding sequence, 3′UTR = 3′ untranslated regionTransfection of siRNA to Cells and mRNA Expression Analysis

LNCaP cells were plated at 3×10⁵ cellsper well on 6 well plates. Thenext day, the cells were transfected with siRNAs (10 nM) usingLipofectamine RNAi Max reagent (Invitrogen, Carlsbad, Calif.) 72 hoursbefore experiments. Total RNA was isolated using ISOGEN reagent (NIPPONGene, Tokyo, Japan). Using 500 ng RNA as template, first-strand cDNA wassynthesized using the Primescript RT reagent kit (TaKaRa Bio) accordingto manufacturer's protocol.

mRNA was quantified by real-time-PCR using the synthesized cDNA (2 μl often times dilution) by KAPA SYBR Fast qPCR Kit (NIPPON Genetics, Tokyo,Japan) and StepOne real-time PCR system (Life technologies, Tokyo,Japan) based on SYBR green fluorescence. The evaluation of relativedifferences of PCR product amounts among the treatment groups wascarried out by the comparative cycle threshold (Ct) method, usingglyceraldehyde-3-phosphate dehydrogenase as an internal control.

The results are shown in FIG. 25. Compared to siControl- orReagent-administered group, PSF mRNA expression decreased insiPSF-administered group. The expression suppression effect of siPSF#1,2, 5, 7, 8 was particularly high.

Cell Proliferation Assay (MTS Assay)

LNCaP Cells were plated at 3×10³ cells per well on 96-well plates andcultured in RPMI-1640 containing 10% FBS. The next day, the cells weretransfected with siRNAs targeting PSF (siPSFs) or negative control siRNAat the concentration mentioned above. 0, 3, and 5 days aftertransfection, MTS assays were carried out using cell titer reagent(Promega Corp., Madison, Wis.) according to the manufacturer's protocol.In short, the number of cells were measured by the dehydrogenasereaction converting a tetrazolium compound (MTS;3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]) by the electron coupling reagent PMS (phenazinemethosulfate) into a formazan product. The quantity of formazan productwas measured by the amount of 490 nm absorbance. The experiments wereperformed in quintuplicate.

The results are shown in FIG. 26. Time-dependent cell growth wasobserved in Reagent- or siControl-administered group. On the other hand,cell growth was suppressed in most siPSF-administered group.

Western Blot Analysis

Whole-cell lysates were prepared using NP40 lysis buffer [50 mM Tris-HCl(pH 8.0), 150 mM NaCl, 1% NP-40, and Protease inhibitor cocktail(Nacalai tesque, Kyoto, Japan)] and resolved in SDS lysis buffer.Lysates were electrophoresised by 8% SDS-PAGE and electroblotted ontoImmobilon-P Transfer Membrane (Millipore Corp., Billerica, Mass.).Membranes were first incubated with primary antibody [anti-PSF (1:1000),anti-β-actin (1:1000)] and then with peroxidase-conjugated antimouse IgGantibody for 1 hour. After extensive washing, the antibodies weredetected using enhanced ImmunoCruz Western blotting detector system(Santa Cruz Biotechnology, Inc).

The results are shown in FIG. 27. The expression of PSF protein wassuppressed by the administration of siPSF#1, #2, #4, #5, #7, and #8.

Prostate Cancer Xenograft Model

Ten million cells in 100 μl PBS with 100 μl of Matrigel (BD Biosciences,San Jose, Calif.) were injected subcutaneously into each side of6-week-old male BALB/c nude mice (Nihon Crea, Tokyo, Japan) using 25 Gsyringe. These mice were untreated until tumors could be observed.

Injection of siRNA into Tumors

When the volume of tumors reached approximately 100 mm³ (i.e. in 4-8weeks after injection), siControl (BannoNegaCon) or siPSF#2 was injectedinto the tumors two times per week (Control group n=5, siPSF group n=6).For the injection, 5 μg siRNA (per mouse) was mixed with 15 μl RNAi MAXin OPTI-MEM were used.

Measurements of Tumor Growth and Evaluation of PSF Expression

Tumors were measured with calipers two times per week. Tumor volume wasdetermined using the formula 0.5×r1×r2×r3 (r1<r2<r3: three axes oftumor). Mice were killed by cervical dislocation 10 weeks after thetransplantation. Animal care was maintained in accordance withinstitutional guidelines. Proteins were extracted by SDS lysis buffer(10 mM Tris-HCl PH7.5, 2% SDS, 10% 2-mercaptoethanol).

The results are shown in FIGS. 28 and 29. The tumor growth in prostatecancer xenograft model was well suppressed by the administration ofsiPSF.

[Sequence Listing Free Text]

SEQ ID NO:1 is a base sequence in one mode of CTBP1-AS.

SEQ ID NO:2 is a base sequence of AX747592.

SEQ ID NO:3 is a base sequence of a target region of siRNA inhibitingthe function of CTBP1-AS.

SEQ ID NO:4 is a base sequence of a target region of siRNA inhibitingthe function of CTBP1-AS.

SEQ ID NO:5 is a base sequence of a target region of siRNA inhibitingthe function of CTBP1-AS.

SEQ ID NO:6 is a base sequence of a target region of siRNA inhibitingthe function of CTBP1-AS.

SEQ ID NO:7 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:8 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:9 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:10 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:11 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:12 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:13 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:14 is a base sequence of RNA constituting siRNA inhibiting thefunction of CTBP1-AS.

SEQ ID NO:15 is a base sequence of RNA constituting siRNA used as acontrol in siRNA in Examples.

SEQ ID NO:16 is a base sequence of RNA constituting siRNA used as acontrol in siRNA in Examples.

SEQ ID NO:17 is a base sequence of CTBP1-ASc.

SEQ ID NO:18 is a base sequence of CTBP1-ASb.

SEQ ID NO:19 is a base sequence of CTBP1-ASa.

SEQ ID NO:20 is a base sequence of CTBP1-ASd.

SEQ ID NO:21 is a base sequence of 3′ Race primer used for 3′ RACE PCR.

SEQ ID NO:22 is a base sequence of a PCR adaptor primer used for 3′ RACEPCR.

SEQ ID NO:23 is a base sequence of a probe used in northern blotting ofRNA derived from LNCaP cells.

SEQ ID NOS:24 to 52 are base sequences of a target region of siRNAinhibiting the function of CTBP1-AS.

SEQ ID NOS:53 to 104 are base sequences of RNA constituting siRNAinhibiting the function of CTBP1-AS.

SEQ ID NOS:105 to 108 are base sequences of a primer used in real timePCR.

SEQ ID NO:109 to 118 are base sequences of a primer used in real timeRT-PCR.

SEQ ID NO:119 is a base sequence of sense strand of siPSF#1.

SEQ ID NO:120 is a base sequence of sense strand of siPSF#2.

SEQ ID NO:121 is a base sequence of sense strand of siPSF#3. SEQ IDNO:122 is a base sequence of sense strand of siPSF#4.

SEQ ID NO:123 is a base sequence of sense strand of siPSF#5.

SEQ ID NO:124 is a base sequence of sense strand of siPSF#6.

SEQ ID NO:125 is a base sequence of sense strand of siPSF#7.

SEQ ID NO:126 is a base sequence of sense strand of siPSF#8.

SEQ ID NO:127 is a base sequence of antisense strand of siPSF#1.

SEQ ID NO:128 is a base sequence of antisense strand of siPSF#2.

SEQ ID NO:129 is a base sequence of antisense strand of siPSF#3.

SEQ ID NO:130 is a base sequence of antisense strand of siPSF#4.

SEQ ID NO:131 is a base sequence of antisense strand of siPSF#5.

SEQ ID NO:132 is a base sequence of antisense strand of siPSF#6.

SEQ ID NO:133 is a base sequence of antisense strand of siPSF#7.

SEQ ID NO:134 is a base sequence of antisense strand of siPSF#8.

The invention claimed is:
 1. A cell growth inhibitor comprising a PSF(PTB-associated splicing factor) expression inhibitor or functioninhibitor as an active ingredient, wherein the PSF expression inhibitoror function inhibitor is a double-stranded RNA which inhibits expressionof PSF, and one strand of the double-stranded RNA comprises a sequencerepresented by any of SEQ ID NO: 121, 122, 123, and
 124. 2. The cellgrowth inhibitor according to claim 1, wherein the cell growth inhibitorinhibits the growth of the cells that express C-terminal binding protein1-antisense (CTBP1-AS) comprising a base sequence of any of SEQ ID NO:17 to
 20. 3. The cell growth inhibitor according to claim 2, wherein thecells that express CTBP1-AS are prostate cancer cells and/or metastaticcancer cells thereof.
 4. A method of treating prostate cancer in asubject, comprising administering a therapeutically effective amount ofthe cell growth inhibitor of claim 1 to the subject.
 5. The cell growthinhibitor according to claim 1, wherein one strand of thedouble-stranded RNA consists of a sequence represented by any of SEQ IDNO: 121, 122, 123, and
 124. 6. A method of treating prostate cancer in asubject, comprising administering a therapeutically effective amount ofthe cell growth inhibitor of claim
 5. 7. The method according to claim4, wherein the administering comprises contacting the cell growthinhibitor of claim 1 to a cell expressing (i) an antisense RNA to aCTBP1 (C-terminal binding protein 1) gene comprising from position 2348to position 2372 of the base sequence represented by SEQ ID NO: 1; or(ii) a mutant or variant of the antisense RNA having a sequence that is90% identical to from position 2348 to position 2372 of the basesequence represented by SEQ ID NO:
 1. 8. The method according to claim4, wherein the cell growth inhibitor inhibits the growth of the cellsthat express C-terminal binding protein 1-antisense (CTBP1-AS),comprising a base sequence of any of SEQ ID NOs: 17 to
 20. 9. The methodaccording to claim 8, wherein the cells that express CTBP1-AS areprostate cancer cells and/or metastatic cancer cells thereof.
 10. Thecell growth inhibitor according to claim 1, wherein the one strand ofthe double-stranded RNA comprises a sequence represented by any of SEQID NO: 121, 123, and 124.